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Research review paper
Plant secondary metabolism linked glycosyltransferases: An update on
expanding knowledge and scopes
Pragya Tiwari
a
, Rajender Singh Sangwan
a,b
,NeelamS.Sangwan
a,
⁎
a
Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Lucknow 226015,India
b
Center of Innovative and Applied Bioprocessing (CIAB), A National Institute under Department of Biotechnology, Government of India, C-127, Phase-8, Industrial Area, S.A.S. Nagar,
Mohali 160071, Punjab, India
abstractarticle info
Article history:
Received 6 October 2015
Received in revised form 6 February 2016
Accepted 19 March 2016
Available online 4 May 2016
The multigene family of enzymes known as glycosyltransferases or popularly knownas GTs catalyze the addition
of carbohydrate moiety to a variety of synthetic as well as natural compounds. Glycosylation of plant secondary
metabolites is an emerging area of research in drug designing and development. The unsurpassing complexity
and diversity among natural products arising due to glycosylation type of alterations including
glycodiversification and glycorandomization are emergingas the promising approaches in pharmacologicalstud-
ies. While, some GTs with broad spectrum of substrate specificity are promising candidates for glycoengineering
while otherswith stringent specificity pose limitationsin accepting molecules and performing catalysis. With the
rising trends in diseases and the efficacy/potential of natural products in their treatment, glycosylation of plant
secondary metabolites constitutes a key mechanism in biogeneration oftheir glycoconjugates possessing medic-
inal properties. The present review highlights the role of glycosyltransferases in plant secondary metabolism
with an overview of their identification strategies, catalytic mechanism and structural studies onplant GTs.Fur-
thermore, the article discusses the biotechnological and biomedical application of GTs ranging from detoxifica-
tion of xenobiotics and hormone homeostasis to the synthesis of glycoconjugates and crop engineering. The
future directions in glycosyltransferase research should focus on the synthesis of bioactive glycoconjugates via
metabolic engineering and manipulation of enzyme's active site leading to improved/desirable catalytic proper-
ties. The multiple advantages of glycosylation in plant secondary metabolomics highlight the increasing signifi-
cance of the GTs, and in near future, the enzyme superfamily may serve as promising path for progress in
expanding drug targets for pharmacophore discovery and development.
© 2016 Published by Elsevier Inc.
Keywords:
Catalytic mechanism
Drug designing
Glycoconjugates
Glycosyltransferases
Metabolic engineering
Plant sec ondary meta bolism
Contents
1. Introduction.............................................................. 715
1.1. Plantsecondarymetaboliteglycosylationmetabolism:anintroduction ................................ 715
2. Plantsecondarymetabolismglycosyltransferases:anoverview ....................................... 717
3. PlantGTsinCAZYdatabase ....................................................... 717
4. IdentificationandisolationofGTs .................................................... 718
4.1. Transcriptomicsand/ormetabolomicsstrategies .......................................... 718
4.2. FunctionalanalysisofUGTgenesthroughclassicalapproaches.................................... 719
4.3. Bioinformaticstudies....................................................... 719
4.4. Biochemicalmethods ...................................................... 719
4.5. Molecularbiologystudies..................................................... 720
5. Kineticparametersofcatalysisandsubstratepreferences.......................................... 720
6. Family1GTs:themechanismofcatalysis................................................. 720
7. Crystalstructureofglycosyltransferases ................................................. 720
Biotechnology Advances 34 (2016) 714–739
Abbreviations: GTs,glycosyltransferases;PSPG, plant secondaryproduct glycosyltransferases; UGT,UDP dependent glycosyltransferase;TOGT, Tobacco-O-glucosyltransferase;ATTED-
II, database of gene coexpression in Arabidopsis;AtUGT72B1, Arabidopsis thaliana UGT72B1; MtUGT71G1, Medicago truncatula UGT71G1; MtUGT85H2, Medicago truncatula UGT85H2;
VvGT1, Vitis vinifera flavonoid 3-O-glucosyltransferase; vvUFGT, Vitis vinifera anthocyanidin glucosyltransferase.
⁎Corresponding author.
E-mail addresses: nss.cimap@gmail.com,sangwan.neelam@gmail.com (N.S. Sangwan).
http://dx.doi.org/10.1016/j.biotechadv.2016.03.006
0734-9750/© 2016 Published by Elsevier Inc.
Contents lists available at ScienceDirect
Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv
7.1. GT-Afamily........................................................... 729
7.2. GT-Bfamily........................................................... 729
8. 3Dstructureofplantglycosyltransferases ................................................ 729
8.1. Flavonoid/triterpene GT from M. truncatula ............................................. 729
8.2. Flavonoid specific glycosyltransferase (WsFGT) from W. somnifera .................................. 729
8.3. Flavonoid glucosyltransferase (CaUGT3) from C. roseus ....................................... 729
9. BiotechnologicalandbiomedicalapplicationsofGTs............................................ 730
9.1. Roleindefensemechanism ................................................... 730
9.2. Synthesisofvaluableglycoconjugates............................................... 730
9.3. GTsinvolvedinhormonalregulation ............................................... 730
9.4. GTs involved in modification of xenobiotics and detoxificationofpollutants.............................. 733
9.5. GTsinvolvedinsecondarymetabolitebiosynthesis......................................... 733
9.6. Stabilizationofsecondarymetabolites............................................... 733
9.7. Plant-microbeinteractions.................................................... 733
9.8. Metabolicengineeringofcrops.................................................. 734
9.9. PharmacologicalstudiesusingUGTs................................................ 734
10. Futureprospectsinglycosyltransferaseresearch............................................. 734
Acknowledgments ............................................................. 735
References................................................................. 735
1. Introduction
Glycosyltransferases (GTs) catalyze stereospecific and regiospecific
transfer of nucleotide diphosphate-activated sugars to a wide and
diverse range of molecules from proteins, lipids, nucleic acids to antibi-
otics and other low molecular weight compounds known as secondary
metabolites (Lairson et al., 2008; Weadge and Palcic, 2009). Glycosylation
mechanism is the key modification step occurring in various biological
processes resulting in formation of myriad of plant secondary metabolites
possessing glycodiversity. Together with hydroxylation, methylation and
acylation reactions, glycosylation contributes to the complexity and di-
versity of plant secondary metabolites. As per the IUBMB guidelines, gly-
cosyltransferases have separate Enzyme Commission numbers and are
classified on various parameters including similarities based on amino
acids (Campbell et al., 1997; Coutinho et al., 2003), substrate specificity,
reaction mechanism (inversion or retention of anomeric carbon)
(Coutinho et al., 2003), 3D structures (GT-A, GT-B or predicted GT-C)
and type of reaction. Till 2015, GTs have been classified into 97 families
(GT1-GT97, (http://www.cazy.org/GlycosylTransferases)withGT-1fam-
ily consisting of maximum candidates of UGT genes. 108 GT crystal struc-
tures are reported in Protein data bank comprising of 40 members
classified in GT-A sub-family, 58 members in GT-B sub-family, 2 mem-
bers in GT-C sub-family and 8 members have remained unclassified
(http://www.rcsb.org/pdb). It is interesting to note that CAZY family
shows a conserved mechanism of catalysis within its members and anal-
ysis of the primary sequence does not provide any information on enzy-
matic function (Breton et al., 2006). GTs have been further sub-divided
into four clans namely‐inverting GT-A fold (clan I) or GT-B fold (clan II)
or retaining GT-A fold (clan III) or GT-B fold (clan IV). The four clans
include members of each CAZY family (Coutinho et al., 2003). (See Fig. 1.)
Past decades have witnessed extensive progress in studies highlight-
ing the significance of GT superfamily but availability of limited
biochemical data on individual member enzyme has hampered further
research for their functional understanding. Recent trends indicate the
identification and biochemical characterization of a number of GT
genes with broad/specific functionality and their analysis at the tran-
scriptome and metabolome levels has made significant contributions
in studying the glycosylation mechanisms in planta. Large scale plant
genome sequencing projects have contributed in deciphering the bio-
logical role of plant secondary metabolism glycosyltransferases in bio-
synthesis of glycoconjugates of phytochemicals (Bhat et al., 2013; Ito
et al., 2014), metabolic engineering of crops (Kristensen et al., 2005;
Lim, 2005a; Weis et al., 2008) as well as prospective role in identifying
key targets for drug designing and pharmacophore development
(Williams et al., 2007). Prior reports have suggested that incorporation
of a sugar moiety to low molecular weight secondary metabolites influ-
ences acceptor's properties like solubility, stability, bioactivity, subcellu-
lar localization and binding properties with other molecules leading to
reduced toxicity of endogenous and exogenous substances (Lim et al.,
2004). For example, terpenoids such as monoterpenols (geraniol and
linalool) are toxic for the plant as such and are chemically hydrophic
thus affecting their mobility and transport across tissues in plants. How-
ever, glycosylation results into the generation of a monoterpenol gluco-
side which becomes transportable, less toxic, stable and also attains
altered volatility affecting aroma (Bonishch et al., 2014).
Glycosyltransferases bear considerable importance and interest
owing to the fact that glycan moiety forms an integral and essential
component of natural products, conferring pharmacological properties
to the molecule leading to enhanced bioavailability, reduced toxicity
and increased solubility. Although, the present trends have highlighted
the significant prospects of GTs in drug-designing anddevelopment, the
stringent specificity of some GTs limits glycodiversification and intro-
duces the need for GT engineering (Williams et al., 2007)whileothers
are promiscuous tools in alterations involving glycosylation patterns.
Emerging trends in glycoengineering have witnessed the manipulation
of active sites in enzymes and site-directed mutagenesis with some suc-
cess (Gutmann and Nidetzky, 2012; He et al., 2006; Modolo et al., 2009).
The present article provides anupdate on the knowledge and scopes of
the glycosyltransferase gene family and its functional aspects, more spe-
cifically in plant secondary metabolism, methodologies employed for
their identification and isolation, multi-faceted role in biotechnological
and biomedical applications and the possible exploration of why the
superfamily may serve as novel targets in drug discovery and develop-
ment in future.
1.1. Plant secondary metabolite glycosylation metabolism: an introduction
Plant secondary metabolites form one of the most important group
of metabolites finding extensive applications in food, nutraceuticals,
medicine and pharmaceutical preparations. Theplant secondary metab-
olism is in theglobal focal attention due to such growingapplications of
its products, and there is a clear uprising trend in studies pertaining to
the pathways and biochemical reactions utilizing and biosynthesizing
plant secondary metabolites. Terpenoidal secondary metabolites are
biosynthesized through a complex network of reactions involving dif-
ferential participation of MVA and DOXP pathways (Akhtar et al.,
2012; Chaurasiya et al., 2012; Narnoliya et al., 2014). The wide variety
of secondary metabolites have immense structural/chemical group di-
versity such as terpenoids, alkaloids, phenylpropanoids, and steroids
and many of them are hydrophic and toxic to the producing cell.
715P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Owing to this, most of them are stored in specific structures (Sharma et
al., 2013; Yadav et al., 2014). Such secondary metabolites undergo var-
ious important terminal transformations catalyzed by specificenzymes
so that their properties are modified in terms of volatility, toxicity and
mobility. Two of the such important transformations are catalyzed by
acyltransferases and glycosyltransferases in plants (Sharma et al.,
2009; Sharma et al., 2014; Tiwari et al., 2014). Acyltransferases are
major contributor in enhancing volatility through esterification where-
as glycosyltransferases modify the properties and nature of aglycones
through glycosylation led lowering of volatility, if at all existed with
the molecule (Sharma et al., 2009; Sharma et al., 2013). In this article,
we have focused on the studies pertaining to glycosyltransferases in-
volved in plant secondary metabolism. Many of the secondary metabo-
lites are hydrophobic in nature and hence mobilized to targeted
localizations within the cell as well as transported to modified struc-
tures such as trichomes (Sangwan et al. 2001; Yadav et al., 2014).
Most of the metabolites are synthesized, transported and accumulated
at secluded structural modifications such as glandular trichomes in
plants (Shanker et al., 1999; Sharma et al., 2013; Bose et al., 2013;
Odimegwu et al., 2013; Yadav et al., 2014). Glucosyltransferases in-
volved in plant secondary metabolism are those set of enzymes which
bring out the addition of glycosyl moiety to hydrophobic
phytoconstituents commonly referred as aglycones. Many of such reac-
tions are highly specific and diverse considering the nature of the
existing phytomolecules in nature (Singh et al., 2014). Such glycosylat-
ed derivatives of secondary metabolites not only exhibit tremendous
structural diversity but also the pharmaceutical and medicinal signifi-
cance. (Singh et al., 2014).
716 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
2. Plant secondary metabolism glycosyltransferases: an overview
The enzymesuperfamily is ubiquitous, accountingfor approximate-
ly 1–2% genes in archaea,bacteria and eukaryotes (Lairson et al., 2008).
Family 1 consists of maximum number of UGT candidates and placed in
clan II. The family 1 GTs exhibits inverting mechanism of catalysis and
depict GT-B structural fold. Low molecular weight substances with
\\OH,\\COOH,\\NH
2
,\\SH and C\\C functional groups are preferred
for the enzymatic catalysis for single or multiple addition of a sugar moi-
ety (Lim and Bowles, 2004). UDP-glucose serves as the primary sugar
donor in family 1 GT followed by UDP-galactose, UDP rhamnose, UDP
xylose and UDP-glucuronic acid respectively (Lim and Bowles, 2004).
The category of sugar acceptors defines a wide range of plant secondary
metabolites namelyterpenes, sterols, phenolics, alkaloids, cyanohydrins
and thiohydroximates (Vogt and Jones, 2000). The enormous diversity
and complexity among the glycosides generated by the catalytic sugar
decoration suggest the presence of a class/family of GTs in plants but
on contrary, it has been seen that plant secondary metabolome defines
the occurrence of GTs.
Considering the structural aspects, a characteristic feature of plant
GTs is the presence of a consensus sequence, the plant secondary meta-
bolic signature sequence (PSPG box), present in secondary metabolism
GTs (Hughes and Hughes, 1994). The PSPG box is regarded as a con-
served domain, essentially present in GTs involved in natural product
glycosylation with only slight modifications and 60–80%sequence iden-
tity (Vogt and Jones, 2000). The N-terminal portion is more variable,
suggesting that the catalytic domain might be involved in binding of di-
verse sugaracceptors. It is interestingto note that family 1 GT is soluble
enzymes as compared to membrane-anchored mammalian GTs. An in
silico motif diversity analysis has shown that the PSPG box was found
to be consistently present in all GT sequences at the C-terminal, discov-
ered through MEME tool. A wide range of sequences was analyzed for
the presence of PSPG motif and phylogenetic evolution of the PSPG
motif characteristic of plant secondary metabolic glycosyltransferases
was studied (Kumar et al., 2012).
The sugar dependent enzymes or Leloir enzymes possess
remarkable functionality in glycosylation of diverse secondary
metabolites resulting in generation of myriad of glycoconjugates
(Tiwari et al., 2014a; Tiwari et al., 2014b). The addition of a carbohy-
drate moiety to secondary metabolites such as flavonoids, terpenoids,
phenolics, saponins, sterols and alkaloids results in positive implications
on their properties thereby influencing their bioactivities. While,
flavonoids constitute the largest family of secondary metabolites
glycosylated by GTs, followed by phenolic GTs and hormone homeosta-
sis GTs, glycoconjugates of certain classes namely alkaloids and
cyanogenic compounds are relatively very few and still need to be
identified.
3. Plant GTs in CAZY database
Carbohydrates are diverse molecules widely present in nature and
perform a range of biological functions from structural components to
carbon deposits and intra- and intercellular cell signaling between
organisms. The Carbohydrate-Active Enzymes (CAZY) database was
made accessable in 1999 and included a comprehensive database
linking the sequence, structure and function of the classified enzymes.
The CAZY database (CAZY, http://afmb.cnrs-mrs.fr/CAZY/) represents
more than 12,000 GT encoding sequences and includes the enzyme
families which create, degrade or modify glycosidic bonds namely bio-
synthesis of sugar conjugated molecules by glycosyltransferases and
degradation by glycosidases, carbohydrate lyases and polysaccharidely-
ases, respectively (Lombard et al., 2014). The database provides online
access to the sequence-based classification of the gene families and
their structural and functional information, upgraded time to time.
Certain salient parameters define the features of the CAZY database
and include the classification of the families based on significant
amino-acid sequence homology, module based classification (CAZymes
are modular proteins) and the analysis of protein sequence released in
GenBank systematically. However, in contrast to other enzymes namely
proteases, esterases, DNAses etc. whose specificity cannot be estimated
from their sequence information, the broad substrate preference of
CAZymes can be predicted from their family classification (Cantarel
et al., 2012). Presently, the database includes genome information of
N2800 genomes classified in the respective kingdoms namely bacteria
(2351), viruses (240), eukaryota (73) and archaea (158), respectively
(Lombard et al., 2014). The methodology for gene sequence analysis
comprises of a variety of methods, tools and aids of bioinformatics in-
cluding their combinations such as a combination of Hidden Markov
Model (HMM) and Blast tools which compares protein models with se-
quence information of catalytic and non-catalytic modules in CAZY da-
tabase, followed by a manual monitoring by experts.
Literature has suggested that a high percentage of plant GTs are in-
volved in glycosylation of small molecules and classified in family 1 of
the CAZY database. The whole-genome sequencing of the model plant,
Fig. 1. Rooted phylogenetic treeconstructed through neighbor joiningmethod of MEGA 5.05software highlighting theevolutionary relationshipof GTs in plant secondary metabolism. The
accession nos. of GTsinvolved in plantsecondary metabolism in increasing orderof evolution areas follows: glycosyltransferase UGT95A1 [Hieracium pilosella] (Accession no. 171906260),
glucosyltransferase-like protein [Crocus sativus] (Accession no. 34015076), UDP-glucose:solanidine glucosyltransferase [Solanum tuberosum] (Accession no. 375004896),
glycosyltransferase [Withania somnifera] (Accession no. 221228775), flavonoid glucosyltransferase [Allium cepa] (Accession no. 32816176), zeatin O-glucosyltransferase 3 [Arabidopsis
thaliana] (Accession no. 46318045), ABA-glucosyltransferase [Vigna angularis] (Accession no. 18151384), ABA glucosyltransferase [Citrus sinensis] (Accession no. 367465462), UDP-glu-
cose: chalcononaringenin 2′-O-glucosyltransferase [Dianthus caryophyllus] (Accession no. 52839682), tetrahydroxychalcone glucosyltransferase [Dianthus caryophyllus] (Accession no.
156138797), anthocyanin 3′-glucosyltransferase [Gentiana triflora] (Accession no. 27530875), phenylpropanoid:glucosyltransferase 2 [Nicotiana tabacum] (Accession no. 13492676), sa-
licylate-induced glucosyltransferase [Nicotiana tabacum] (Accession no. 1685005), cyanohydrin UDP-glucosyltransferase UGT85K5 [Manihot esculenta] (Accession no. 346682867), UDP-
glycosyltransferase 85C1 [Stevia rebaudiana] (Accession no. 37993673), UDP-glucose anthocyanin 5-Oglucosyltransferase [Medicago truncatula] (Accession no. 355499268), UDP-glucose
glucosyltransferase [Rhodiolasachalinensis] (Accession no. 145280639), UDP-xylose phenolic glycosyltransferase[Solanum lycopersicum] (Accession no. 350534960), UDP-glycosyltrans-
ferase BMGT1 [Bacopa monnieri] (Accession no. 302310821), UDP-glucoseglucosyltransferase [Fragaria ×ananassa](Accession no. 51705411), glucosyltransferase [Vitis vinifera](Acces-
sion no. 363805188), limonoid UDP glucosyltransferase [Citrus maxima] (Accession no. 160690854), limonoid UDP glucosyltransferase [Citrus aurantium] (Accession no. 160690840),
hydroxycinnamate glucosyltransferase[Brassica napus] (Accession no.88999675), sinapate1-glucosyltransferase [Brassica oleracea var. medullosa] (Accession no. 226533664), flavonoid
glucosyltransferase [Crocus sativus] (Accession no. 222646154), monoterpene glucosyltransferase [Eucalyptus perriniana] (Accession no. 60650093), UDP-glucose:anthocyanin 5-O-
glucosyltransferase [Perilla frutescens var. crispa] (Accession no. 4115559), UDP-glucose:flavonol 5-O-glucosyltransferase homolog [Solanum melongena] (Accession no. 112806966),
DIMBOA UDP-glucosyltransferase BX9 [Zea mays] (Accession no. 226505740), UDP-glucose: anthocyanidin 3-O-glucosyltransferase [Freesia hybrid cultivar] (Accession no.
301353154), UDP glucose:flavonoid 3-O-glucosyltransferase [Ipomoea trifida](Accession no. 32441911), UDP glucose-flavonoid 3-O-glucosyltransferase [Malus ×domestica] (Accession
no. 329790853), flavonoid 3-glucosyltransferase [Rosa hybrid cultivar] (Accession no. 327343824), flavonoid 3-O-glycosyltransferase [Litchi chinensis] (Accession no. 309951616),
UDP-glucose:flavonoid-O-glucosyltransferase [Beta vulgaris] (Accession no. 46430997), glucosyltransferase [Phytolacca americana] (Accession no. 219566994), glycosyltransferase
[Panax notoginseng] (Accession no. 332071130), UDP-glucose glucosyltransferase [Gardenia jasminoides] (Accession no. 342306020), tetrahydroxychalcone 2′-glucosyltransferase
[Catharanthus roseus] (Accession no. 156138819), UDP-glucose:glucosyltransferase[Cucumis melo subsp.melo] (Accessionno. 307136362), chalcone 4′-O-glucosyltransferase[Antirrhinum
majus] (Accessionno. 379067424),UDP-glucuronate:baicalein7-O-glucuronosyltransferase [Scutellaria baicalensis] (Accessionno. 37359710),UGT2 [Pueraria montanavar. lobata](Acces-
sion no. 216296852), UDP-glycose:flavonoid glycosyltransferase [Vigna mungo] (Accession no. 4115536), zeatin O-xylosyltransferase [Phaseolus vulgaris] (Accession no. 5802783), puta-
tive UDP-glucose:flavonoid glucosyltransferase [Ginkgo biloba] (Accession no. 378829085), flavonoid glycosyltransferase UGT94C2 [Veronica persica] (Accession no. 260279128), UDP-
glucose:sesaminol 2′-O-glucoside-O-glucosyltransferase [Sesamum indicum] (Accession no. 165972256), UDP-glucose:flavonoid 3-O-glucosyltransferase [Prunus persica](Accessionno.
339715876), UDP-rhamnose:soyasaponin III-rhamnosyltransferase [Glycine max] (Accession no. 292684225), UDP rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase
[Petunia ×hybrida](Accessionno.397567).
717P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Arabidopsis thaliana have led to the comprehensive analysis of the GT
superfamily in this plant. Sequence analysis methods have identified
the presence of 120 GT sequences, grouped in family 1 in Arabidopsis ge-
nome. The identified genes in the Arabidopsis genome have the C-
terminal consensus and classified as UGT's with three exceptions
(Paquette et al., 2003). The family 1 GTs are further divided into two
subsets: one subset consists of a PSPG conserved domain for nucleotide
sugar binding, present as well in mammalian GTs which glycosylate in-
ternal metabolites like steroids, dietary flavonoids and xenobiotics (Lim
and Bowles, 2004). The other subset is relatively smaller, consisting of
GTs catalyzing sterol and glycerolipid glycosylation (Warnecke et al.,
1997). Microbial GTs leading to antibiotic biosynthesis namely
urdamycin (Hoffmeister et al., 2001), vancomycin (Zmijewski and
Briggs, 1989) and vicenistatin (Ogasawara et al., 2004) are classified
together in family 1 GTs. The glycosylation modification enhances the
hydrophilicity and stability of small molecules thereby influencing
their bioactivities (Thorson et al., 2001). Moreover, low molecular
weight compounds are the substrate for family 1 GTs with the function-
al groups \\COOH, \\NH
2
,\\OH, C\\Cand\\SH functional groups
present on substrate acting as probable glycosylation sites (Bowles et
al., 2006). The structural information on plant GTs is important to
decode the functional mechanisms and their evolution, however the
availability of very few crystal structures of plant GTs namely
flavonoid/triterpene glycosyltransferase (UGT) from Medicago
truncatula (Shao et al., 2005), flavonoid glycosyltransferase (WsFGT)
from Withania somnifera (Jadhav et al., 2012)andflavonoid
glucosyltransferase (CaUGT3) from Catharanthus roseus (Masada et al.,
2009)defines the major limitation. The structural information reveals
that plant GTs contain two Rossmann folds, where the activated donor
sugar binds in the C-terminal region whereas the acceptor binds in the
N-terminal region of the protein. The members of family 1 GTs partici-
pate in diverse functions in plant secondary metabolism and play an
important role in the growth and development of the plant (Wang
and Hou, 2009).
4. Identification and isolation of GTs
The breakthrough in whole genome sequencing and high-
throughput screening and analysis of sequences have revolutionalized
the investigations on glycosyltransferases in recent years. It is believed
that plant glycosyltransferases are most abundant in nature as com-
pared to other organisms, considering the complex polysaccharide na-
ture of cell wall and glycosylation of secondary metabolites in plants
(Hansen et al., 2010). Various experimental methodologies in areas of
molecular biology, bioinformatics, biochemistry, DNA sequencing of ge-
nomes/transcriptomes, genome-wide association studies (GWAS) and
quantitative trait locus (QTL) mapping have facilitated the isolation,
cloning and analysis of genes encoding plant GTs. Earlier strategies in
isolation of these genes involved classical genetic approaches and bio-
chemical studies such as isolation of mutants (Dooner and Nelson,
1977) and enzyme purification (Vogt and Jones, 2000) which forms
the underlying platform in GT identification and research. Molecular bi-
ology techniques have facilitated the isolation and cloning of GT genes
from microbes and plants into heterologous systems. Other identifica-
tion methods for UGT genes included homology-basedscreening of con-
served domains or homologous genes for cDNA library screening
(Martin et al., 2001a, 2001b). Additionally, biochemistry has
revolutionalized the entire GT research as the uncharacterized genes
and the newly discovered ones are being increasingly characterized
and their catalytic role in plants is being investigated and established.
Other identification methods included the use of combinational genetic
libraries to establish N-linked glycosylation in yeast (Choi et al., 2003),
use of web-based software for screening GT candidates SEARCHGTr
(Kamra et al., 2005), development of rice databases for identification
of GTs from rice (Cao et al., 2008) and glycogene database in Japan
(JCGG-DB, http://riodb.ibase.aist.go.jp/rcmg/ggdb), utilization of
glycoproteomic techniques namely Lectin-IGOT-LC/MS (an LC–MS
based approach) for β-1,4-galactosyltransferase-I (b4GalT-I), an iso-
zyme of glycosyltransferase (Sugahara et al., 2012), Glycogene microar-
rays for studying differential expression of glycosyltransferases in
corneas of mice (Saravanan et al., 2010), network-based approach to
predict novel GTs (Sánchez-Rodríguez et al., 2014) and employing met-
abolic quantitative trait loci (mQTL) analysis for the determination of
secondary metabolism associated genomic regions in fruit pericarp of
tomato (Alseekh et al., 2015).
Furthermore, quantitative genetic approaches together with regula-
tory network analysis were used for the identification of several key
genes in plant secondary metabolism in Zea mays (Wen et al., 2016).
The variation in plantsecondary metabolites and theunderlying genetic
constitution have been analyzed employing metabolic quantitative trait
locus (QTL) studies. In one such study, the genetically inbred lines of
two populations were employed to determine the natural variation in
kernels of Z. mays. Furthermore, 57 QTL's were validated through ge-
nome wide association studies (Wen et al., 2014) and a gene regulatory
network was established for flavonoid biosynthetic pathway. Several
products of the respective genes were identified, a putative flavonol-
3-O-glucosyltransferase (for vitexin biosynthesis) been one of them
(Wen et al., 2016).
Glycogenomics, a mass spectrometry-guided genome-mining meth-
od comprises of a prospective approach for determination of microbial
glycosylated molecules. The method was employed for characterization
of glycosylated natural products and their biosynthetic network from
sequenced genome of microbes. The tandem mass spectrometry (MS)
technique was used to characterize N and O glycosyl group in the
sugar monomers and similarityto the respective secondary metabolism
gene was established through MS-glycogenetic code. Furthermore, the
genes involved in aglycone biosynthesis (GNP) are classified (Kersten
et al., 2013). In addition to these, some other identification strategies
are discussed in details for the identification and isolation of candidate
genes involved in glycosylation of plant secondary metabolism.
4.1. Transcriptomics and/or metabolomics strategies
Large scale whole genome sequencing projects in the present era
have been a breakthrough in the identification of new UGTs in
A. thaliana. The decoding of complete genome sequence of A. thaliana,
the model plant has revealed the presence of the glycosyltransferase
multigene family subject to initial analysis. The genome elucidation of
Arabidopsis showed the presence of 100 putative GTs involved in the
glycosylation changes in plant secondary metabolites (Li et al., 2001).
A superfamily of 119 UGTs was identified in Arabidopsis genome with
the PSPG motif, the signature sequence conserved in members of family
1 GT respectively. The sequencing of whole plant genomes had uncov-
ered a huge family of UGT genes with novel functions. Transcriptome-
based strategies like transcriptome coexpression analysis, is a promising
technique in the identification of genes pertaining to secondary
metabolism in plants. A set of regulatory genes regulate the activities
of genes operating in a specific metabolic pathway and coexpressed in
a specific organ or in a defined environmental condition (Yonekura-
Sakakibara, 2009).
Flavonoids, the class of secondary metaboliteshave been extensively
studied for their glycosylation mechanism. The coexpression database
ATTED-II (http://atted.jp) serves as a major resource for identification
of new flavonoid encoding GTs in plants. Among 107 candidates in
Arabidopsis,five UGTs were found to correlate with flavonoid biosyn-
thetic genes with highest correlation with UGT89C1, for example two
flavonoid UGTs were known namely flavonoid 3-O-glucosyltransferase
and anthocyanin 5-O-glucosyltransferase (Tohge et al., 2005).
Recently, genome-wide identification and expression profiling stud-
ies in developmental tissue stages revealed the presence of 96 UGT
genes in Cicer arietinum genome. The analysis further highlighted the
differential expression pattern such as 84 CaUGTs showed high
718 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
expression levels while 12 CaUGTs revealed low levels of expression
(Sharma et al., 2014). A transcriptome coexpression and independent
component analysis was undertaken to identify candidate genes in
Arabidopsis utilizing 1388 microarray data available in public database.
A. thaliana, a model plant extensively studied for flavonoid metabolism
and UGT79B1 and UGT84A2 were identified as anthocyanin
glucosyltransferases. Out of the two genes, UGT79B1 exhibited similar
expression and correlation with anthocyanin biosynthetic genes while
UGT84A2 did not show significant correlation and was related to flavo-
nol biosynthetic genes, respectively (Yonekura-Sakakibara et al., 2012).
Transcriptomic analysis was performed for the identification of MeJA-
inducible genes in Panax ginseng (Jung et al., 2014). The study constitut-
ed the sequencing and de novo assembly of P. ginseng transcriptome and
two GTs namely PgUGT74AE2 and PgUGT94Q2 were characterized and
studied for their functional role in ginsenoside biosynthesis (Jung et al.,
2014).
4.2. Functional analysis of UGT genes through classical approaches
Classical approaches like cloning, purification of protein, mutant
isolation and cDNA libraries screening using heterologous probes have
been used to identify the functions of UGTs (Vogt and Jones, 2000).
The catalytic activity of UGTs was determined by heterologous expres-
sion in Escherichia coli or yeast and in vitro enzymatic assays. This has re-
vealed that UGTs had broad substrate specificity in vitro (Hansen et al.,
2003). The enzyme is selective for a particular or few positions on the
substrate (Lim et al., 2004). A technique to study the subcellular locali-
zation of the enzyme involved green fluorescent protein (GFP), fusion
proteins showed that UGT85B1 constitutes complex of multienzymes
that biosynthesizes dhurrin, a cyanogenic glucoside (Kristensen et al.,
2005). Recently, an up and down regulation strategy of gene expression
was used to determine the regulation of scopoletin levels by TOGT, phy-
toalexin accumulation subject to tobacco mosaic virus infection. More-
over, oxidative stress in plants was found to increase with
downregulation ofTOGT indicated that accumulation of scopoletin is re-
sponsible for deactivation of oxygen formed due to oxidative stress
(Chong et al., 2002; Gachon et al., 2004).
4.3. Bioinformatic studies
The progress in the area of bioinformatics made a significant contri-
bution in the identification of plant glycosyltransferases. Conserved
motifs were discovered through the use of various motif discovery
tools which showed that PSPG box is a conserved consensus sequence
found in most of the GTs involved in glycosylation of secondary metab-
olites in plants. This was a good beginning in the identification of new
GTs from a database. While some methods in GT identification utilized
motif search programs (Kikuchi and Narimatsu, 2006) others adopted
BLAST approach to mine out new GTs (Cantarel et al., 2009; Campbell
et al., 1997). In 2006, under Glycogene project, Kikuchi and Narimatsu
developed a bioinformatic system for the identification and in silico
cloning of human glycogens. The study identified and engineered 105
glycogenes corresponding to human for heterologous expression and
38 recombinant proteins were characterized for substrate specificity.
The bioinformatic system comprised of multiple strategies including in
silico cloning through Phrap and GENSCAN, using profile Hidden
Markov Model for clustering glucosyltransferases and deciphering
glycosyltransferase evolution (Kikuchi and Narimatsu, 2006).
In silico tools determine the related expressed sequence tags (ESTs),
cDNA or genes and molecular biology methods such as mining of com-
plete open reading frames of genes, their expression in heterologous
system (E.coli or yeast) and biochemical techniques like enzyme purifi-
cation and characterization are sequentially performed. Several GTs for
plant hormones namely auxins, cytokinins, brassinosteroids (BR) and
abscisic acid (ABA) were identified through these methods (Hou et al.,
2004; Poppenberger et al., 2005). A study by Hansen and colleagues
(2010) used a combination of multiple bioinformatic methods to iden-
tify new putative glycosyltransferases in A. thaliana.Theyusedseveral
remote homology detection methods namely Profile Hidden Markov
Models (HMM), Hydrophobic Cluster Analysis (HCA), BLAST and PSI-
BLAST and 3D-Fold Recognition and discovered more than 150 protein
sequences corresponding to glucosyltransferase class of enzymes. An
example includes the accession ID At5g03795.1 which was discovered
by employing all the three bioinformatic approaches and showed simi-
larity to exostosin protein family. The N-terminal domain catalyzing ad-
dition of β-1, 4-glucuronic acid residues is classified in GT47 family
while the C-terminal domain introduce α1,4-N-acetylglucosaminyl res-
idues to the acceptor is classified in GT64 family. Moreover, the study
highlighted the presence of GT signature sequence in DUF266 and
DUF246 protein families although distantly related to CAZY families
GT14 and GT65, respectively. The data generated helped in the annota-
tion of Arabidopsis genome, however the characterization of the identi-
fied genes accounts for the major challenge in glycobiology (Hansen
et al., 2008).
Additionally, to identify new GTs not reported in CAZY database,
multiple bioinformatic tools were developed. Transmembrane Hidden
Markov Model (TMHMM) 2.0 prediction server (Krogh et al., 2001)
was employed for screening of Arabidopsis proteome (26,095 protein
sequences) and identified 27 putative GT sequences. About 90% of the
protein sequences were filtered and 2611 sequences were submitted
to the SUPERFAMILY prediction server, a selection method used for pre-
diction of GT related superfamilies. The probable sequences were run
through 3D-PSSM (Kelley et al., 2000) and GenTHREADER (McGuffin
and Jones, 2003) (fold-recognition) servers. Another in silico strategy
depended on the increasing availability and elucidation of 3dimensional
structures of GTs, utilizing the remote homology detection methods
(HMMer program (Eddy, 1998), PSI-BLAST and Structural overlap
(Sov) parameter calculations) (Geourjon et al., 2001). Chemometrics
and bioinformatics define powerful methods to analyze whole genomes
(identify GTs in Mycobacterium tuberculosis genome) (Wimmerova
et al., 2003). Principal Component Analysis (PCA) forms an important
statistical tool for analysis of data in large collection of data.
4.4. Biochemical methods
Earlier, purification of the target enzyme, determination of substrate
preferentiality and enzyme kinetics were the few available methods to
identify and categorize new GTs. With the advent of high-throughput
technologies and discovery of novel GTs in plant genomes,the biochem-
ical characterization of the enzymes forms an integral aspect in
glycobiology. Studies have made substantial contribution in delineating
the functional role in glucosylation of plant secondary metabolites
through characterization of the large repertoire of available GTs in
public domain. With large complexity in enzymatic functions and
discovery of the broadas well as substrate-specific GT genes, the utiliza-
tion of biochemical techniques in glycobiology constitutes an integral
approach in identification and validation of the uncharacterized
collection of GT clones. Wei et al. (2015) isolated four novel GTs namely
UGTPg100, UGTPg101, UGTPg102, and UGTPg103 (Yan et al., 2014)and
established the catalytic activities of the corresponding enzymes. The
metabolic pathway of ginsenosides (pharmacological value) biosynthe-
sis was elucidated through the synthesis of glycon gensenosides of
protopanaxatriol (PPT) and PPT-type ginsenosides. Another study
reported the purification of a UDP-glucose anthocyanin 3′-O-
glucosyltransferase from Gentiana triflora functionally involved in mod-
ification of flower color (blue) and anthocyanin biosynthesis (Fukuchi-
Mizutani et al., 2003). Similar study by Ogata et al. (2001) reported the
purification of an anthocyanin 5-O-glucosyltransferase from the flowers
of Dahlia variabilis and studied its role in the biosynthesis of aliphatic
acylated anthocyanins in flower petals, respectively. Furthermore,
biochemical methods employed for GT identification led to the
isolation of key GT enzymes like a 2,4,5-trichlorophenol detoxifying O-
719P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
glucosyltransferase from Triticum aestivum (Brazier et al., 2003), a UDP-
glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) from G. triflora
involved in isoflavone glucosylation (Noguchi et al., 2007), UDP-
glucose:p-hydroxymandelonitrile-O-glucosyltransferase from Sorghum
bicolor catalyzing the last step in cyanogenic glucoside (dhurrin) bio-
synthesis (Jones et al., 1999), detoxification of the pollutant 3,4-
dichloroaniline by GT72B1 from A. thaliana (Loutre et al., 2003), BX8
and BX9 catalyzing detoxification of benzoxazinoids in maize (Rad
et al., 2001) to name a few.
However, some challenges also exist in employing the biochemical
strategies for GT identification. These methods have limitation since
many GTs are present in very minute quantities. GTs are very labile
and occur in relatively low quantitative abundance which make the pu-
rification of the enzyme difficult. GTs in secondary metabolism, are sol-
uble enzymes generally with a molecular mass between 45 kDa and
60 kDa. A combination of techniques including anion exchange, dye li-
gand chromatography and hydrophobic interactions (Vogt and Jones,
2000)isusedforproteinpurification.
4.5. Molecular biology studies
Molecular biology techniques like polymerase chain reaction (PCR),
cDNA cloning and heterologous expression of genes have opened new
avenues in the identification and characterization of GT enzymes.
Several enzymes involved in glucosylation of anthocyanidins and
flavonoids from various plant sources were identified by cDNA library
screening and using GT probes for differentially expressed cDNAs. The
data obtained have greatly enhanced knowledge of natural
product GTs and emphasize on acceptor specificity. For example
p-hydroxymandelonitrile GT also accepts mandelonitrile (struc-
tural resemblance) and benzyl alcohol, exhibiting broad spectrum
activity. The enzyme also shows activity for monoterpenoid geraniol
as for benzyl alcohol (Jones et al., 1999). The salicylic acid glucosylating
GT shows enzymatic preference for acyl group of p-hydroxybenzoic
acid or benzoic acid compared to endogenous substrates (Lee and
Raskin, 1999). These examples show the specificity of GTs for individual
hydroxyl groups.
Several studies on GTs functionally involved in biosynthesis of
secondary metabolites of therapeutic/food value are increasingly
discovered and explored for its commercial value. The complexity and
diversity of secondary products are enormously being valued for phar-
macological significance, stress responses, detoxification of pollutants,
plant-microbe interactions, hormone regulation or pathway engineer-
ing to name a few. The use of homology based PCR screening approach
has led to the identification of several GT genes namely a sterol GT
which catalyzes glucosylation of cholesterol (present in bacteria) and
ergosterol (fungus) to stigmasterol (plants) thus paving way for
glycol-engineering of sterol metabolism (Tiwari et al., 2014a, 2014b),
GT from Ipomea nil and its ability to glucosylate different phytohor-
mones (Suzuki et al., 2007), defense responses to stress conditions
(Lim and Bowles, 2004; Kanoh et al., 2014), detoxification of pesticides
and herbicides (Wetzel and Sandermann, 1994), enhanced bioavailabil-
ity (Thorson et al., 2001), detoxification of xenobiotics (Brazier et al.,
2007) and UGT94F4 and UGT86C4, involved in the biosynthesis of
picroside, an iridoid glycoside possessing pharmacological properties
(Bhat et al., 2013). The methods in molecular biology offers a practical
approach to delineate the functional role of glucosyltransferase in
plant secondary metabolism.
5. Kinetic parameters of catalysis and substrate preferences
A vast set of plant secondary metabolic glycosyltransferases has
been characterized with respect to their biochemical and kinetic prop-
erties. New researches on GTs have added to our knowledge on enzyme
activity and its catalytic mechanisms. The proteomic characterization of
the enzyme elucidates the actual biochemical parameters influencing
enzyme's function and shed light on their physiological role in plants.
Several studies have enriched the biochemical data on GTs based on
their substrate preferentiality, regioselectivity, their catalytic properties
as well as structural elucidation. Focused researches have significantly
contributed in transcriptome analysis and indicated that there might
be several GTs and not one which might be present in a system.Further-
more, interpretation of GTs at the molecular level employing structural
and kinetic studies is significant for understanding the functional prop-
erties of enzymes enacting as drug targets in diseases (Gloster, 2014).
Delineating enzyme kinetics withrespect to V
max
,K
m
and K
cat
as param-
eters of kinetics of their catalysis for a defined substrate would reveal
the biochemical characteristics of theenzyme and would further estab-
lish the biosynthetic/catalytic role of GTs in plant. Table 1 summarizes
the literature available on biochemical and kinetic characterization of
GTs functionally involved in glycosylation of plant secondary
metabolites.
6. Family 1 GTs: the mechanism of catalysis
Glycosyltransferase, the biocatalysts demonstrate natural course of
evolution for catalyzing glycosylation reaction. Many GTs (microbial)
find application in antibiotic glycosides and oligosaccharides biosynthe-
sis (Koizumi et al., 1998; Mendez and Salas, 2001) but the use of plant
GTs as biocatalyst was limited due to unavailability of plant GT
sequences (Lim et al., 2005). The identification of several plant GTs,
recombinant expression of the protein and catalytic characterization
have greatly enhanced our knowledge about the catalytic mechanism
of these enzymes. GTs which catalyze the glycosylation of plant second-
ary metabolites are classified in family 1and designated as GTs for small
molecules (Henrissat and Coutinho, 2001; Li et al., 2001). In plants,
cytosolic UGTs glycosylate a wide array of natural products such as
terpenoids, flavonoids, phenylpropanoids, terpenoids and steroids
(Bowles et al., 2006).
Plant GTs have been foundto glycosylate both aglyconeand glycone
molecules. Some members of family 1 catalyze the sugar transfer to
glycosides while some plant GTs glycosylate the aglycone moiety of
secondary metabolites, for example formation of quercetin-3,7-di-O-
glucoside (Lim et al., 2004). GTs classified in family 1 catalyze the O, S-
as well as N-linkages and can form different linkages with the particular
substrate. For example, Arabidopsis GT 72B1 forms an N-glucosidic bond
with 3, 4-dichloroaniline (a pollutant) and an O-glucosidic bond with 3,
4-dihydroxybenzoic acid (Loutre et al., 2003; Xu et al., 2013). GTs which
are phylogenetically related display regioselectivity but employ differ-
ent sugar donors for glycosidic linkage formation. GTs add sugar resi-
dues to diverse nucleophile acceptors namely carbon, oxygen,
nitrogen and sulfur and except C-glycosylation GTs, most GTs are in-
volved in heteroatom glycosylation (O-glycosylation, N-glycosylation,
and S-glycosylation) respectively (Chang et al., 2011). Examples include
A. thaliana UGT72B1, which catalyzes O-glycosylation, N-glycosylation,
and S-glycosylation (Chang et al., 2011). Recently, a bi-functional C
and O glucosyltransferase was discovered from Z. mays catalyzing
glycosylation of flavonoid C and O glucosides (Ferreyra et al., 2013).
7. Crystal structure of glycosyltransferases
Although, in recent years, a number of GTs have been isolated from
different sources but problems associated with their overexpression,
purification and crystallization have made the studies on crystal struc-
ture difficult. The first crystal structure was reported in 1994 for T4-
glucosyltransferase, a bacteriophage (Vrielink et al., 1994). Information
on GT structure is available for 17 distinct GT families consisting of both
inverting and retaining enzymes, available at Glyco3D site (http://
www.cermav.cnrs.fr/glyco3d). Furthermore, the structural information
on GTs obtained in the last 4 years has provided important information
on GTs, their enzymatic mechanism of action and specificity (Gloster,
2014).
720 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Table 1
Kinetic parameters of catalysis and substrate preferences of plant secondary metabolic glycosyltransferases.
GT/plant system NCBI/Genbank/DDBJ/Uniprot
accession number
K
m
V
max
Substrate Products Reference
UGT74AC1 from Siraitia grosvenorii HQ259620 41.4 μM
58.2 μM
54.7 μM
–Mogrol
Quercetin
Naringenin
Mogroside IE
Quercetin glycosides
Naringenin glycosides
Dai et al. (2015)
UGT85K11 and UGT94P1 from Camellia
sinensis
AB847092
AB847093
UGT85K11
Geraniol
44.2 μM
332.1 nkat mg
−1
protein
Monoterpene
Aromatic
Aliphatic alcohols
β-primeverosides Ohgami et al. (2015)
UGTPg1, UGTPg100, UGTPg101,
UGTPg102 and UGTPg103 from
Panax ginseng
KP795113 KP795114
KP795115
KP795116
104 μM
153 μM
386
426
Protopanaxadiol
Protopanaxatriol
Ginsenosides Wei et al. (2015)
UGT80B1 from
Arabidopsis thaliana
At1g43620 –– β-sitosterol Sitosterol glycoside Mishra et al. (2015)
UGT84A17 from
Populus trichocarpa
DY801582 –– Caffeic acid
4-coumaric acid
4-hydroxybenzoic acid
2-coumaric acid
Ferulic acid
Sinapic acid
Caffeoyl-glucose
4-coumaroyl-glucose
4-hydroxybenzoyl-glucose
Feruloyl-glucose
Cinnamoyl-glucose
Benzoyl-glucose
Babst et al. (2014)
UGT74S1, UGT74T1, UGT89B3,
UGT94H1, UGT712B1 from
Linum usitatissimum L.
JX011632
JX011633
JX011634
JX011635
JX011636
–– Secoisolariciresinol Secoisolariciresinol
monoglucoside
Secoisolariciresinol
diglucoside
Ghose et al. (2014)
UGT708C1 and UGT708C2 from
Fagopyrum esculentum M.
AB909375
AB909376
–– 2-hydroxyflavanones
2-hydroxynaringenin
2-hydroxyeriodictyol
2-hydroxypinocembrin
Dihydrochalcone (phloretin)
Trihydroxyacetophenones
2-hydroxynaringenin C-glucoside
2-hydroxyeriodictyol C-glucoside
2-hydroxypinocembrin C-glucoside
Phloretin C-glucoside
Trihydroxyacetophenones
Nagatomo et al.
(2014)
SrUGT74G1 from
Stevia rebaudiana
AY345982.1 –– Steviolbioside Stevioside Guleria and Yadav
(2014)
PgUGT74AE2 and PgUGT94Q2 from
Panax ginseng
JX898529
JX898530
25 μM–Protopanaxadiol
Protopanaxatriol
Ginsenoside Rg3 and Rd Jung et al. (2014)
TaUGT4 from
Triticum aestivum
tplb0040h20 –– Deoxynivalenol Deoxynivalenol 3-glucoside Xin et al. (2014)
GsSGT from
Gymnema sylvestre R.Br.
NS –– Sterols
Cholesterol
Ergosterol
Stigmasterol
Steryl glucosides
Cholesteryl β-D glucoside
Ergosteryl β-D glucoside
Stigmasterol β-D glucoside
Tiwari et al. (2014b)
PNgt1 and PNgt2 from Pharbitis nil AB757750
AB757751
–– Flavonoids
Umbelliferone
3-hydroxy flavone
3,6-dihydroxy flavone
3,7-dihydroxy flavone
Benzaldehyde derivatives
Vanillin
Vanillyl alcohol
Coumarins
Scopoletin
Esculetin
Skimmin Kanoh et al. (2014)
AdGT4 from
Actinidia deliciosa
KF954944 –– Terpene alcohol/alcohol
aglycones
Hexanol
(Z)-hex-3-enol
Terpene glycosides Yauk et al. (2014))
GhSGT1 and GhSGT2 from Gossypium
hirsutum
JN004107
JN004108
15.1 μM
12.6 μM
0.56 pmol mg
−1
min
−1
0.90
β-sitosterol β-sitosterol glucoside Li et al. (2014)
UDP-glycosyltransferases from BRADI1G43600 –– Deoxynivalenol Deoxynivalenol-3-O-glucoside Schweiger et al.
(continued on next page)
721P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Table 1 (continued)
GT/plant system NCBI/Genbank/DDBJ/Uniprot
accession number
K
m
V
max
Substrate Products Reference
Brachypodium distachyon (2013)
UGT6, UGT7 and UGT8 from
Catharanthus roseus
AB591741 UGT6
NA
0.088 μM
0.202 μM
1.99 μM
7-Deoxyloganetic acid
7-Deoxyloganetin
7-deoxyloganic acid
7-deoxyloganin
Asada et al. (2013)
KF411463 –– – –
WsGT from
Withania somnifera
FJ560880 8.488 μM
9.335 μM
11.61 μM
12.79 μM
13.01 μM
37.79
39.79
9.89
9.885
6.961
Flavonoid-7-ols
Diadzein
Naringenin
Genistein
Luteolin
Apigenin
Diadzein 7-O-glucoside
Naringenin 7-O-glucoside
Genistein 7-O-glucoside
Luteolin 7-O-glucoside
Apigenin 7-O-glucoside
Singh et al. (2013)
NSGT1 from
Solanum lycopersicum
KC696865 –– Phenylpropanoid volatiles-V diglycosides
Eugenol
Guaiacol
Methyl salicylate
PhP-V triglycosides
Eugenol-2-O-b-D-glucopyranosyl-(1 →
2)-[O-b-D-xylopyranosyl-(1 →6)]-O-b-D-glucopyranoside (GXG)
Guaiacol-GXG
Methyl Salicylate-GXG
Tikunov et al. (2013)
UGT94F2 and UGT86C4 from Picrorhiza
kurrooa
JQ996408 JQ996409 –– – Picrosides Bhat et al. (2013))
UGT73C11 and UGT73C13 from
Barbarea vulgaris
JQ291614
JQ291616
UGT73C11
9.7 μM
3.3 μM
UGT73C13
12.5 μM
22.9 μM
817 nmol min
−1
mg
−1
390
231
131
Aglycone sapogenins
Oleanolic acid
Hederagenin
Oleanolic acid
Hederagenin
3-O-β-D-Glc oleanolic acid
3-O-β-D-Glc hederagenin
Augustin et al. (2012)
UGT75L6 and UGT94E5 from
Gardenia jasminoides
AB555731
AB555739
UGT94E5
0.072 mM
0.023 mM
–Crocetin Crocin-5, crocetin-mono-(b-glucosyl)-ester
Crocin-3, crocetin-di-(b-glucosyl)-ester
Nagatoshi et al. (2012)
UGT71A15 from
Malus x domestica
DQ103712 82 μM
188 μM
243 μM
244 μM
354 μM
8μmol/s kg
54
49
79
85
Phloretin
Kaempferol
Quercetin
Phloridzin (phloretin 2′-O-glucoside)
7-O-glucoside of kaempferol
3-O-glucoside of kaempferol
7-O-glucoside of quercetin
3-O-glucoside of quercetin
Gosch et al. (2012)
UGTSr from
Stevia rebaudiana
–––Stevioside Rebaudioside A Madhav et al. (2012)
UGT79B1 and UGT84A2 from
Arabidopsis thaliana
AB018115
AB019232
–– Cyanidin 3-O-glucoside
Sinapic acid
Cyanidin 3-O-xylosyl(1 →2)glucoside
1-O-sinapoylglucose
Yonekura-Sakakibara
et al. (2012)
UGT707B1 from
Crocus sativus
HE793682 –– Kaempferol
Quercetin
Kaempferol-3-O-b-D-Glucopyranosyl-(1–2)-β-D-Glucopyranoside
Quercetin-3-O-rhamnosyl(1 →2)-glucoside-7-O-rhamnoside
Trapero et al. (2012)
sgtl3.1, sgtl3.2 and sgtl3.3 from
Withania somnifera
EU342379
EU342374
EU342375
–– Sterols Sterol glucoside Chaturvedi et al.
(2012)
–––– –
UGT72B14 and UGT74R1 from
Rhodiola sachalinensis A. Bor.
EU567325
EF508689
UGT72B14
4.7 μM
UGT74R1
172.4 μM
57.8 pkat mg
−1
293.1
Tyrosol Salidroside (tyrosol 8-O-b-D-glucoside) Yu et al. (2011)
UGT85A24 from
Gardenia jasminoides
AB555732 8.8 mM
0.61 mM
–Genipin
7-deoxyloganetin
Loganetin
Geniposide
Gardenoside
7-deoxyloganetin
1-O-glucoside
Loganetin 1-O-glucoside
Nagatoshi et al. (2011)
UGT85K4 and UGT85K5 from Manihot
esculenta
JF727883
JF727884
–– Acetone cyanohydrin
2-hydroxy-2-methylbutyronitrile
Cyanogenic glucosides Linamarin
Lotaustralin
Kannangara et al.
(2011)
SlUGT5 from
Solanum lycopersicum
HM209439 –22.1 nkat/mg
−1
protein
19.8
7.62
Methyl salicylate
Guaiacol
Eugenol
Methyl salicylate glucoside
Guaiacol glucoside
Eugenol glucoside
Benzyl alcohol glucoside
Louveau et al., 2011
722 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
4.43
–
121.3
77.5
Benzyl alcohol
Phenyl ethanol
Hydroquinone
Salicyl alcohol
Not Detected
Hydroquinone glucoside
β-isosalicin
TaGTa-TaGTd from Triticum aestivum
ScGT from Secale cereale
AB547237
AB547238
AB547239
AB547240
–– Benzoxazinones
2,4-dihydroxy-1,4-benzoxazin-3-one
2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one
DIBOA glucose
DIMBOA glucose
Sue et al. (2011))
F3GT1 and F3GGT1 from Actinidia
chinensis
GU079683
FG404013
–– Anthocyanidin
Cyanidin
–Montefiori et al.
(2011)
THC2’GT from Carnation,Cyclamen and
Catharanthus roseus
–––Tetrahydroxychalcone 4,2′,4′,6′-Tetrahydroxychalcone (THC) 2′-glucoside Togami et al. (2011)
UGT76B1 from
Arabidopsis thaliana
AC008153 –– Isoleucic acid Isoleucic acid glucoside Paul et al. (2011)
PlUGT1 from
Pueraria lobata
EU889119 –– Daidzein Diadzin Zhou et al. (2011)
VvgGT1, VvgGT2 and VvgGT3 from
Vitis vinifera
JN164679
JN164680 JN164681
–– Hydroxybenzoic acids (C6C1)
4-hydroxybenzoic acid
Protocatechuic acid
Gallic acid
Syringic acid
Hydroxycinnamic acids (C6C3)
p-coumaric acid
Caffeic acid
Sinapic acid
Stilbene
Trans-resveratrol
Flavonoids
Quercetin
Cyanidin
Catechin
p-Hydroxybenzoyl-D-glucose
Protocatechoyl-D-glucose
Galloyl-D- glucose
Syringoyl-D- glucose
p-Coumaroyl-D-glucose
Caffeoyl-D- glucose
Sinapoyl-D- glucose
NA
NA
NA
NA
Khater et al. (2011)
UGT78K1 from
Glycine max (L.) Merr.
GU434274 174 μM
16 μM
24.8 pKat μg
−1
–
Anthocyanidins and flavonol aglycones
Kaempferol
Cyanidin
Kaempferol 3-O-glucoside
Cyanidin 3-O-glucoside
Kovinich et al.
(2010)
ScUGT1-ScUGT5 from Sinningia
cardinalis
AB537178
AB537179
AB537180
AB537181
AB537182
145.8 μM
633.2 μM
60.2 nmole mg
−1
min
−1
635.3
Flavonoids
Apigeninidin
Lutcolinidin
Apigeninidin 5-O-glucoside
Luteolinidin 5-O-glucoside
Nakatsuka and
Nishihara (2010)
UGT88D8 from
Veronica persica
AB465708 10.72 μM–Apigenin –Ono et al. (2010b)
CsUGT1, CsUGT2 and CsUGT3 from
Citrus sinensis L.Osbeck
GQ221686
GQ221687
GQ221688
–– Terpenoids Terpenoid glycosides Fan et al. (2010)
SbUGT from
Scutellaria barbata
GU339042 –– Flavonol
Kaempferol
Flavanone
Naringenin
Flavone
Apigenin
Isoflavones
Daidzein
Kaempferol 7-O-glucoside
Kaempferol 3-O-glucoside
Naringenin 7-O-glucoside
Apigenin 7-O-glucoside
Diadzein 7-O-glucoside
Chiou et al. (2010)
GmSGT2 and GmSGT3 from Glycine max AB473730
AB473731
–– – Soyasapogenol B Shibuya et al. (2010)
UDP-GT from Catharanthus roseus –0.112 mM
0.077 mM
0.064 mM
1.0 mM
–
–Scopoletin
5,7-dihydroxyflavone
5,7-dihydroxyflavanone
–
–
UDPG
Umbelliferone
Isoscopoletin
Scopoletin 7-O-b-monoglucoside
Dihydroxyflavone 5-O-b-monoglucoside
Dihydroxyflavanone 7-O-b-monoglucoside
Umbelliferone 7-O-b-monoglucoside
Isoscopoletin 6-O-b-monoglucoside
Esculetin 6-O-b-monoglucoside
Piovan et al. (2010)
(continued on next page)
723P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Table 1 (continued)
GT/plant system NCBI/Genbank/DDBJ/Uniprot
accession number
K
m
V
max
Substrate Products Reference
Esculetin
CaUGT3, CaUGT4 and CaUGT5 from
Catharanthus roseus
AB443870
AB443871
AB443872
CaUGT3
0.38 μM
0.36 μM
0.69 μM
2.00 μM
0.14 μM
0.18 μM
1.11 μM
0.32 μM
N2.50 μM
0.41 μM
0.67 μM
0.58 μM
N2.50 μM
N2.50 μM
1.95 μM
N2.50 μM
N2.50 μM
0.72 μM
F
lavonol glycosides
Quercetin 3-O-glucoside
Kaempferol 3-O-glucoside
Myricetin 3-O-glucoside
Quercetin 3-O-gentiobioside
Flavone glycosides
Apigenin 7-O-glucoside
Luteolin 7-O-glucoside
Flavanone glucoside
Liquiritigenin 4′-O-glucoside
Isoflavone glucoside
Genistein 7-O-glucoside
Cyanidin 3-O-glucoside
Curcumin glycosides
Curcumin monoglucoside
Curcumin diglucoside
Curcumin gentiobioside
Coumarin
Esculin
Phenolics
Arbutin
p–Nitrophenylglucoside
Purnasin
Salicin
UDP-glucose
Quercetin 3- O -gentiobioside
Quercetin 3-O-gentiotrioside
Quercetin 3-O-gentiotetroside
Masada et al. (2009)
PaGT 1, PaGT2 and PaGT 3 from
Phytolacca americana L.
AB458516
AB458515
AB458517
PaGT3
145 μM
20 μM
66 μM
20 μM
–
Capsaicin
Quercetin
Apigenin
Genistein
–Noguchi et al. (2009)
UGT90A7, UGT95A1 and UGT72B11
from
Hieracium pilosella L.
EU561019
EU561020
EU561016
UGT90A7
9.5 μM
11.8 μM
UGT95A1
46.8 μM
UGT72B11
20.2 μM
4.7 pkat/μg
4.0
27.2
7.6
Luteolin
Eriodictyol
Luteolin
Kaempferol
Luteolin 3′-O-glucoside
Eriodictyol 3′-O-glucoside
Luteolin 3′-O-glucoside
Kaempferol 3-O-glucoside
Kaempferol 7-O-glucoside
Witte et al. (2009)
UGT85H2 from
Medicago truncatula
DQ875463 35.5 μM
118.9 μM
4.1 μM min
−1
mg
prot
4.0
Flavonol
Kaempferol
Isoflavone
Biochanin A
Kaempferol 3-O-glucoside
Biochanin A 7-O-glucoside
Modolo et al. (2009)
CsGT45 from
Crocus sativus
FJ194947 15.6 μM
86.95 μM
30.3 μM
21.50 μM
366 pkat/mg
186
22.9
104
Kaempferol 7-OH
Quercetin 4′-OH
Quercetin 3′-OH
Quercetin 7-OH
Kaempferol 7-O-glucoside
Quercetin 7-O-glucoside Quercetin 4′-O-glucoside
Quercetin 3′-O-glucoside
Moraga et al. (2009)
OsCGT from
Oryza sativa ssp. Indica
FM179712 16.5 μM
–
–
–
2.5 μM
NA
–
–
–
–
–2,5,7-trihydroxyflavanone
2-Hydroxyflavanone
2,5-Dihydroxyflavanone
2,7-Dihydroxyflavanone
2-Hydroxynaringenin
2-Hydroxyeriodictyol
Flavones
Chrysin
Apigenin
Luteolin
Naringenin
2,5,7-hydroxyflavanone-C-glucoside
2-hydroxyflavanone-C-glucosides
2-hydroxyflavanone-O-glucosides
2,5-Dihydroxyflavanone C-glucosides
2,7-Dihydroxyflavanone C-glucosides
2-Hydroxynaringenin C-glucosides
2-Hydroxyeriodictyol C-glucosides
Chrysin 6-C-glucosides
Chrysin 8-C-glucosides
Apigenin C-glucosides
Luteolin C-glucosides
Brazier et al. (2009)
724 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
8.3 μM
4.78 μM
8.0 μM
–
–
–
Naringenin chalcone
2′,4′,6′-Trihydroxydihydrochalcone
Phoretin
2,4,6-Trihydroxybenzophenone
Maclurin
1,3,5-Trihydroxybenzoic acid
1,3,5-Trihydroxyacetophenone
Benzyl 2,4,6-trihydroxybenzoate
Naringenin C-glucosides
Naringenin chalcone C-glucosides
2′,4′,6′-Trihydroxydihydrochalcone
Phoretin C-glucosides
Maclurin C-glucosides
1,3,5-Trihydroxybenzoic acid C-glucosides
1,3,5-Trihydroxyacetophenone C-glucosides
Benzyl 2,4,6-trihydroxybenzoate C-glucosides
UGT73F2 from
Glycine max
DQ278439 28.33 μM
6.3 μM
164.37 μM
–Daidzein
Glycitein
Genistein
Daidzin
Glycitin
Genistin
Dhaubhadel et al.
(2008)
UDP-glucose: sterol GT from Solanum
melongena
–– Steroid alkaloids
a) Spirosolane type
Tomatidine
Solasodine
b) Solanidane type
Solanidine
Demissidine
Steroid sapogenin
Nuatigenin
Isonuatigenin
Hecogenin
Diosgenin
Tigogenin
Sarsasapogenin
Yamogenin
Sterols and their derivatives
Sitosterol
Stigmasterol
Stigmasta-5,24(28)-dien-3β-ol
Stigmastan-3β-ol
Cholesterol
25-Hydroxycholesterol
Cholest-5-en-3β,20α-diol
Cholest-5-en-3β,19-diol
Cholest-5-en-3β-ol-7-on
22-Oxycholesterol
5α-Cholest-7-en-3β-ol
Thiocholesterol
5α-Cholestan-3β-ol
5β-Cholestan-3β-ol
5α-Cholestan-3α-ol
5β-Cholestan-3α-ol
Androstane or pregnane derivatives
Androstenolon
Pregnenolon
Triterpenic alcohols
Lanosterol
β-Amyrin
Steryl glucosides Potocka and
Zimowski (2008)
FaGT1 from
Fragaria x ananassa
AAU09442 30 μM 21 nkat/mg Anthocyanidins
Pelargonidin
Cyanidin
Peonidin
Kaempferol
Quercetin
Delphinidin
Petunidin
Malvidin
Flavonol
Galangin
Fisetin
Isorhamnetin
Pelargonidin 3-O-glucoside
Cyanidin 3-O-glucoside
Peonidin 3-O-glucoside
Kaempferol 3-O-glucoside
Quercetin 3-O-glucoside
Griesser et al.
(2008a, 2008b)
(continued on next page)
725P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Table 1 (continued)
GT/plant system NCBI/Genbank/DDBJ/Uniprot
accession number
K
m
V
max
Substrate Products Reference
Myricetin
UGT73B4 and UGT73C1 from
Arabidopsis thaliana
AC006248
AC006282
UGT73B4
0.95 mM
0.44
–
0.15
0.29
4.36 nkat mg
−1
3.32
–
0.008
0.021
2-hydroxylaminodinitrotoluene
4-hydroxylaminodinitrotoluene
2-aminodinitrotoluene
4-aminodinitrotoluene
2-HADNT-O-monoglucoside
4-HADNT-O-monoglucoside
Gandia-Herrero et al.
(2008)
Gt5GT7 from
Gentiana triflora
AB363839 29.5 mM
20.9 mM
12.2 mM
3.4
NA
NA
NA
1.49 nmol min
−1
mg protein
−1
0.98
0.68
0.52
Delphinidin
Cyanidin
Pelargonidin
Malvidin
Kaempferol
Apigenin
Naringenin
Delphinidin 3’O glucoside
Cyanidin 3’O glucoside
Pelargonidin 3’O glucoside
Malvidin 3’O glucoside
Nakatsuka et al.
(2008)
UGT706C1, UGT706D1, UGT707A3 and
UGT709A4 from
Oryza sativa
AP003560
AP003560
AP005186
AP005190
–– Apigenin
Daidzein
Genistein
Kaempferol
Luteolin
Naringenin
Quercetin
Apigenin-7-O-glucoside
Diadzein 7-O-glucoside
Genistein 7-O-glucoside
Kaempferol 3-O-glucoside
Luteolin 3′-O-glucoside
Luteolin 4′-O-glucoside
Luteolin 7-O-glucoside
Naringenin 7-O-glucoside
Quercetin 3-O-glucoside
Ko et al. (2008)
GmIF7GT from
Glycine max
AB292164 3.6 μM–Genistein
Daidzein
Formononetin
Quercetin
Kaempferol
4,2′-tetrahydoxychalcone
4′,6′-tetrahydoxychalcone
Apigenin
Aureusidin
Esculetin
Naringenin
Genistein 7-O-β-D-glucopyranoside
(genistin)
Daidzein 7-O-glucoside
Formononetin 7-O-glucoside
Quercetin 7-O-glucoside
Kaempferol 7-O-glucoside
4,2′-tetrahydoxychalcone 7-O-glucoside
4′,6′-tetrahydoxychalcone 7-O-glucoside
Apigenin 7-O-glucoside
Aureusidin 6-O-glucoside
Esculetin 7-O-glucoside
Naringenin 7-O-glucoside
Noguchi et al. (2007)
SGTL1, SGTL2 and SGTL3 from
Withania somnifera
DQ356887
DQ356888
DQ356889
40 μM
10
25
10
6
27
40
4
4.5
7
ND
8pmol mg
−1
min
−1
7.2
2.5
1.8
0.6
0.8
0.6
0.8
0.2
0.2
0.5
ND
Dehydroepiandrosterone
Deacetyl 16-DPA
Transandrosterone
3-b-hydroxy 16,17- α
epoxypregnenolene
Pregnenolene
Stigmasterol
β-sitosterol
Ergosterol
Brassicasterol
Solasodine
5-α-chol estan-3-β-ol
Cholesterol
Sterol glucosdes Sharma et al. (2007)
27β-hydroxy GT from Withania
somnifera
–0.17 mM
0.11
0.11
0.01
0.05
0.17
0.04
0.07
0.08 pmol/min
0.05
0.04
0.02
0.03
1.05
0.15
0.06
17α-OH withaferin A
27β-OH withanone
Withanolide A
5α,6β,17α,27β-
Tetrahydroxywithanolide
Withanolide U
Testosterone
Estradiol
21β-OH progesterone
–Madina et al. (2007)
UGT85H2 from DQ875463 2.9 μM 0.1073 μM min
−1
Kaempferol Kaempferol 3-O-glucosde Li et al. (2007)
726 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Medicago truncatula 2.8 μM
4.8 μM
1918 μM
9.9 μM
0.0184
0.0257
0.0124
0.0742
Quercetin
Biochanin A
Genistein
Isoliquiritigenin
Quercetin 3-O-glucosde
Biochanin A 7-O-glucoside
Genistein 7-O-glucoside
–
UGT74M1 from
Saponaria vaccaria
DQ915168 170 μM
51 μM
42 μM
37 μM
–Gypsogenic acid
16-OH gypsogenic acid
Gypsogenin
Quillaic acid
Gypsogenin 28-glucoside Meesapyodsuk et al.
(2007)
UGT84A10 and UGT84A11 from
Brassica napus
AM231594
AM231595
–– Hydroxycinnamates
Ferulate
Sinapate
4-coumarate
Cinnamates
Cinnamate
Caffeate
β-acetal esters Mittasch et al.
(2007)
PsUGT1 from
Pisum sativum
–––Kaempferol
Quercetin
Apigenin
Taxifolin
Naringenin
Indole Acetic Acid
Cytokinin
Kaempferol-C-glucuronide
Quercetin-C-glucuronide
Apigenin-C-glucuronide
Taxifolin-C-glucuronide
NA
NA
NA
Woo et al. (2007)
RUGT-10 from
Oryza sativa
AP006584 –– Flavanone
Naringenin
Flavone
Apigenin
Flavonol
Kaempferol
Naringenin 7-O-glucoside
Apigenin 7-O-glucoside Apigenin 4′-O-glucoside
Kaempferol 3-O-glucoside Kamepferol 7-O-glucoside
Kamepferol 4′-O-glucoside
Hong et al. (2007))
FaGT2 from
Fragaria ananassa
AY663785 356.9 μM
603.5 μM
707.7 μM
358.5 μM
315.7 μM
300.2 μM
502.6 μM
464.4 μM
642.4 μM
515.3 μM
108.0 μM
411.2 μM
431 μM
488.2 μM
2.34 nkat mg
−1
2.69
2.59
1.65
2.24
1.21
1.08
1.05
0.77
1.69
0.68
0.17
0.22
0.75
Cinnamic acid
p-Coumaric acid
Caffeic acid
Ferulic acid
5-Hydroxyferulic acid
Sinapic acid
Benzoic acid
3-Hydroxybenzoic acid
para-Hydroxybenzoic acid
Vanillic acid
3,4-Dimethoxycinnamic acid
Phenylpropionic acid
Phenylbutyric acid
3-Aminobenzoic acid
Cinnamoyl –D-glucose
p-coumaroyl-D- glucose
Caffeoyl –D-glucose
Feruloyl-D-glucose
5-Hydroxyferuloyl-Dglucose
Sinapoyl-D-glucose
Benzoyl-D-glucose
3-Hydroxybenzoyl-D-glucose
p-Hydroxybenzoyl-Dglucose
Vanillin-D-glucose
Phenylpropionyl-D-glucose
Phenylbutyoyl-D-glucose
3-Aminobenzoyl-D-glucose
4-Aminobenzoyl-D-glucose
Lunkenbein et al.
(2006)
(continued on next page)
727P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Table 1 (continued)
GT/plant system NCBI/Genbank/DDBJ/Uniprot
accession number
K
m
V
max
Substrate Products Reference
437.1 μM 1.73 4-Aminobenzoic acid
RuGT5 from Oryza sativa –239.5 μM
327 μM
120.7 μM
1666.7 pkat/mg
2000
733.3
Kaempferol
Apigenin
Genistein
–Ko et al. (2006)
UGT73A4 and UGT71F1from
Beta vulgaris
AY526080
AY526081
UGT73A4
11.0 μM
19.2 μM
333 μM
UGT71F1
2.4 μM
26.8 μM
29.0 μM
16.4 pkat μg
−1
12.8
0.2
0.7
0.1
0.03
Quercetin
Apigenin
Betanidin
Quercetin
Apigenin
Betanidin
–Isayenkova et al.
(2006)
UGT75L4 and UGT88A4 from
Maclura pomifera
DQ985179
DQ985176
UGT75L4
20.51 μM
23.05 μM
2.99 μM
93.69 μM
59.33 μM
–Dihydrokaempferol
Dihydroquercetin
Kaempferol
Genistein
Isoliquiritigenin
–Tian et al. (2006)
AtGT-1 from
Arabidopsis thaliana
AY14269 18.0 μM
26.3 μM
35.3 μM
–
–
–
333.3 pkat/mg
333.3
250.0
–
–
–
Eriodictyol
Kaempferol
Quercetin
Naringenin
Apigenin
Luteolin
Eriodictyol 7-O-glucoside
Kaempferol 3-O-glucoside
Quercetin 3-O-glucoside
Naringenin 7-O-glucoside
Apigenin 7-O-glucoside
Luteolin 7-O-glucoside
Kim et al. (2006a, b)
Sgt1 and Sgt2 from Solanum tuberosum DQ218276 –– Solanaceous aglycones
Solanidine
α-chaconine
α-solanine
McCue et al. (2006)
UGT73K1 and UGT71G1 from
Medicago truncatula
AY747626
AY747627
GT029H
25 μM
32 μM
45 μM
166 μM
GT049F
165 μM
235 μM
–
Quercetin
Genistein
Biochanin A
Hederagenin
Soyasapogenol B
Hederagenin
Quercetin glycosides
Genistein 7-O-glucoside
Biochanin A 7-O-glucoside
Hederagenin 3- or 28-O-glucoside
Soyasapogenol B 3-, 22-,
or 23-O-glucoside
Hederagenin 3- or 28-O-glucoside
Achnine et al. (2005)
UGT85B1 from Sorghum bicolor AF199453 –– p-hydroxymandelonitrile Cyanogenic glucoside
dhurrin
Thorsoe et al. (2005)
Ih3GT from Iris hollandica AB161175 –– Anthocyanidins
Delphinidin
Malvidin
Cyanidin
Peonidin /Pelargonidin
Delphinidin 3 glucoside
Malvidin 3 glucoside
Cyanidin 3 glucoside
Peonidin 3 glucoside
Pelargonidin 3 glucoside
Yoshihara et al.
(2005)
ND—Not detected.
NA—No activity.
NS—Not submitted.
728 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
GTs had been classified into two structural superfamilies, GT-A and
GT-B, initially reported in SpsA and β-glucosyltransferase (BGT) struc-
tures, respectively (Charnock and Davies, 1999; Vrielink et al., 1994).
In 2008, a third family of GTs was reported. The crystal structure of
GT-C enzyme: STT3 from the archaea Pyrococcus furiosus was
established (Igura et al., 2008). The structure consists of a C-terminal
soluble domain and central core domain including WWDYG motif
responsible for its catalytic activity. The GT folds comprise of α/β/α
sandwich pattern similar to the Rossmann type fold (six-stranded par-
allel β-sheet with 321,456 topology) present in many nucleotide bind-
ing proteins (Lesk, 1995). Till date, four crystal structures of plant GTs
are available namely AtUGT72B1, MtUGT71G1 and MtUGT85H2 and
VvGT1. While, the first three belongs to family 72, 71 and 85, VvGT1
has not been nomenclature according to the classification (Mackenzie
et al., 1997). The four crystallized structures of GTs show 20–35%
amino acid identity and remarkable conservation in their secondary
and tertiary structures (Osmani et al., 2009).
7.1. GT-A family
The GT-A structure consists of α/β/αsandwich, similar to Rossmann
fold and consists of a seven stranded β-sheet (with 3,214,657 topology
in which strand 6 is antiparallel to the rest).The β-sheet in the center is
flanked by a smaller one and both are associated to form an active site
(Breton et al., 2006). The presence of DxD motif and a divalent cation
is a conserved featureof members in GT-A family, essential component
for enzymatic function (Breton et al., 1998; Breton and Imberty, 1999).
7.2. GT-B family
The GT-B structure comprises of two separate Rossmann domains
with a connecting linker region and a catalytic site present between
the domains. The structure of the GT-B family had shown remarkable
conservation mainly in the C-terminal domain which is the nucleotide
binding domain of the enzyme. Structural variations have been found
in the N-terminal domains, in the helices and loops and active site
which evolved to accommodate different acceptors (Breton et al.,
2006). A peptide motif, a glutamate residue and glycine-rich loops
which interacts with the ribose and phosphate moieties of nucleotide
donor, respectively is present in members of GT-B family (Wrabl and
Grishin, 2001).
8. 3D structure of plant glycosyltransferases
The structural information is an important tool to understand the
evolutionary trends and catalytic mechanism of the proteins. Despite
great progress in isolation and characterization of plant GTs, only a
few crystal structures of plant GTs are available. Shao et al. (2005) and
He et al. (2006) presented the crystal structures of a flavonoid/
triterpene glycosyltransferase (UGT) from M. truncatula. It consists of
two Rossmann folds and acceptors bind the residues in the N-terminal
portion of the protein whereas the activated donor sugars bind to
amino acid residues in the C-terminal region. The present trends in GT
research highlight the importance of X-ray crystallographic studies in
elucidation of 3D structure of plant GTs with an aim to unravel thestruc-
tural complexity and utilization of bioinformatic strategies to decode
the enzyme functionality through protein modeling and active site
docking studies. Recent studies on protein modeling and active site ma-
nipulations of GTs are described in Table 2.
8.1. Flavonoid/triterpene GT from M. truncatula
UGT85H2 from M. truncatula, involved in glycosylation of
isoflavonoid class of secondary metabolites was cloned by Li et al.
(2007). Further, crystal structure of UGT85H2 was deduced at 2.1 Å res-
olution which revealed new structural insights about the enzyme and
its function and substrate specificities. Substrate docking, kinetics and
mutational studies decoded the complex structure of the enzyme and
its regiospecificity in terms of preference for different isoflavonoids.
Heterologous expression in E.coli and kinetic characterization showed
that UGT85H2 glucosylated diverse flavonoids namely the isoflavones
genistein and biochanin A, flavonols kaempferol and quercetin, and
the chalcone isoliquiritigenin utilizing UDP-glucose as sugar donor (Li
et al., 2007). 7-O-positions in biochanin A and genistein are preferred
for regiospecific glucosylation as well as for kaempferol, producing
their respective 3-O-glucosides.
As compared to other plant GTs (VvGT1 and UGT71G1), linker re-
gion in UGT85H2 is longer and includes several other insertions and de-
letions. A long insertion with sevenor six residues more is seenbetween
Nα5andNα5a in the N-terminal region. In the C-terminal domain five
and four residue insertions are presentbetweenCβ2andCα2, and also,
a deletion between Cβ6andCα6 is present. Further, Nβ2 is followed by
Nα2 directly with the absence of a loop linker region similar to VvGT1
but different from UGT71G1. Several disordered regions were also
found. These flexible regions differ in their length and conformations
and are present around cleft formed by the N and C-terminal domains
of the enzymes. This likely explains the substrate binding and specificity
and the flexibility might be essential for recognition of different/diverse
acceptors. The conformational changes occurringduring substrate bind-
ing and glycosylation dueto flexible regions in enzyme's active site offer
a possible explanation. The study showed that the presence of histidine
at 21st position and aspartic acid at 125th position is crucial and impor-
tant for the enzymatic activity of UGT85H2 enzyme (Li et al., 2007).
8.2. Flavonoid specific glycosyltransferase (WsFGT) from W. somnifera
3D protein modeling and ligand docking studies have been reported
for a flavonoid glycosyltransferase from W. somnifera by Jadhav et al.
(2012). A protein model was generated for WsFGT through homology
modelingapproach using MODELLER 9v9 and the model wassubjected
to loop refinement and energy minimization through various bioinfor-
matic tools. Further, the protein model showed a significant homology
to M. truncatula UDP-glucuronosyl/UDPglucosyltransferase (2PQ6: 31%
identity) and the docking studies were performed with various flavo-
noid acceptors such as luteolin, diadzein, apigenin, naringenin, genistein
and kaempferol and UDP-glucose as sugar donor. The binding pocket of
the model consists of 13 amino acid residues and these interacted with
substrates with hydrogen bond formation. The sugar donor, UDP-
glucose was completely buried in the C-terminal domainof the enzyme
while the residues involved in the interaction of enzyme with UDP-
glucose, were present in PSPG motif (Jadhav et al., 2012).
8.3. Flavonoid glucosyltransferase (CaUGT3) from C. roseus
A unique glucosyltransferase catalyzing the 1,6-glucosylation of
flavonol and flavone glucosides was isolated from suspension culture
of C. roseus. The functional properties of the enzyme were investigated
through homology modeling and site-directed mutagenesis. A
homology model of CaUGT3 docked with UDP-glucose and quercetin
3-O-glucoside was generated. Vitis vinifera flavonoid 3-O-
glucosyltransferase, VvGT1, crystallized with an acceptor substrate
kaempferol, was used as template. CaUGT3 model has GT-B fold confor-
mation and includes Nand C terminal domains which form a deep cleft
which site for binding of the sugar donor and sugar acceptor substrates.
The docking energy of the model was determined to be
−32.84 kcal mol
−1
. Further, it was assumed that replacement of
Phe121and Phe200 in VvGT1 to His125 and Asn206 in CaUGT3 is
responsible for broad acceptability for substrates. The point mutation
in His
125
Phe and Asn
206
Phe showed that His
125
plays a more important
role as compared to Asn
206
in binding with the acceptor substrates
(Masada et al., 2009).
729P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
9. Biotechnological and biomedical applications of GTs
Considerable work has been carried out on GTs and the availability
of genomic and biochemical data has made important contributions in
biological studies of plant glycosyltransferases. Techniques involving
functional genomics, gene knock-outs/silencing, overexpression of
genes, bioinformatic approaches including protein docking and site-
directed mutagenesis provide a good insight into the physiological
roles of GTs in plants. This indicates that glycosyltransferases might
play an important role in plant growth, development, defense responses
and interaction with the environment.
The major classes of secondary metabolites such as the
phenylpropanoids, flavonoids and benzoates in plants and hormones
namely auxins, gibberellins, cytokinins and abscisic acid occur as glyco-
sides (Kleczkowski and Schell, 1995; Ostrowski and Jakubowska, 2014).
Plant UGTs play a significant role in defense responses to stress condi-
tions (Lim and Bowles, 2004; Kanoh et al., 2014), detoxification of pesti-
cides and herbicides (Wetzel and Sandermann, 1994), enhanced
bioavailability (Thorson et al., 2001), detoxification of xenobiotics (Bra-
zier-Hicks et al., 2007; Messner et al., 2003) biosynthesis (Bhat et al.,
2013), storage and transport of secondary metabolites (Gachon et al.,
2005), hormone homeostasis and synthesis of bioactive natural prod-
ucts (Lim and Bowles, 2004; Paquette et al., 2003; Asada et al., 2013;
Ito et al., 2014). Glycosylation is an important regulatory mechanism
which plays a key role in maintaining cellular homeostasis. A wide
range of sugar moieties is added either independently (monoglycosides)
or in chains (di- or tri-glycosides), resulting in a broad category of glyco-
sides possessing glycodiversity among them (Jones et al., 1999). Studies
have reported that more than 6000 different glycoconjugates of flavo-
noids occur in plants (Anderson and Markham, 2006).
9.1. Role in defense mechanism
The addition of a carbohydrate moiety to a toxic substance converts
it to non-reactive and stable form which can be stored within the cell.
Further, the attachment of a sugar residue would limit the interaction
with other cellular components thereby reducing the chances of
electron transfer from the aglycone to other components resulting
in lower reactivity and thus, better stability of the molecule. Several
examples show the importance of secondary metabolism specific
glycosylation subjecting to defense responses. For example, the to-
bacco glycosyltransferases, TOGTsglycosylateshydroxycoumarin,
scopoletin, and hydroxycinnamic acids. Furthermore, decline in
scopoletin glucoside levels and impaired resistance to Tobacco Mo-
saic Virus (TMV) were observed with downregulation of TOGTs in
the transgenic tobacco (Chong et al., 2002). These findings suggested
that glycosylation of scopoletin by TOGTs increases plant resistance
to pathogens.
Glycosylation of saponins is another remarkable example of defense
mechanism. Saponins are triterpene glycosides in which the oligosac-
charidechain consists of glucose, galactose, arabinopyranose, GlcUA, xy-
lose or rhamnose attached to the C-3 of saponins which may be crucial
for resistance against fungal pathogens. Removal or alteration in the po-
sition of sugar residues leads to loss of bioactivity. It is interesting to
note that fungal pathogen produces hydrolases which attack the C3
chain to detoxify saponins. For instance, Gaeumannomyces graminis,
the oat root infecting pathogen synthesize avenacinase, (a β-
glucosidase) for detoxification of avenicin, (Wang and Hou, 2009). In
another study, Kanoh et al. (2014) reported the isolation and biochem-
ical characterization of PNgt1 and PNgt2, GTs from the hairy roots of
Pharbitis nil. The biochemical characterization of the recombinant pro-
tein showed significant glycosylation for coumarins and benzaldehyde
derivatives, suggesting a correlation with skimmin biosynthesis,
highlighting the significance of plant GTs in defense responses and
phytoalexin production respectively (Kanoh et al., 2014).
9.2. Synthesis of valuable glycoconjugates
Plant glycosyltransferases are involved in in vitro biosynthesis of
glycoconjugates having diverse properties. The addition of sugar moie-
ties to aglycones or to glycones in chains leads to generation of broad
spectrum of secondary/specialized metabolites with unique properties.
For instance, quercetin, a flavonol occurs in 300 different glycosidic
forms in plants. Glycosylation is usually the terminal modification step
in the biosynthesis of secondary metabolites, for example in case of
W. somnifera, sterol glucosyltransferases were found to catalyze the
glucosylation of sterol, probably leading to its stabilization and storage
(Sharma et al., 2007; Madina et al., 2007). Such enzymate catalyzed
synthesis of natural product glycosides are often preferred over the syn-
thetic processes using chemical methods as synthesis of stereospecific
glycosides does not involve the use of chemicals as blocking and
deblocking reagents, with low cost and fewer synthetic steps and the
microbial whole-cell systemscould be used for production in fermenta-
tion reaction at a much larger levels. Therefore, the application of GTs in
synthesis of glycoconjugates has biotechnological relevance (Wang and
Hou, 2009). Recent research by Bhat et al. (2013) identified and charac-
terized UGT94F4 and UGT86C4, involved in the biosynthesis of
picroside, an iridoid glycoside possessing pharmacological properties
(Bhat et al., 2013). The biochemical characterization of GTs in near fu-
ture and establishment of their catalytic properties would facilitate
their application as biocatalysts. Similarly, a C-glucoside specificGT
was cloned from Fagopyrum esculentum, demonstrating catalytic effi-
ciency for flavonoids and THAP-like compounds in E.coli cultures sug-
gesting the possibilities of efficient biotransformation yielding
glucosylated products of therapeutic value (Ito et al., 2014). Other im-
portant applications of GTs include the use of purified enzymes in fer-
mentors and enzyme immobilization to recycle enzymes ((Dulik and
Fenselau, 1988), in plants employing whole cell systems by internal
GTs and supplementation of exogenous aglycones. The main advantage
is that the activated sugar donor for the reaction is provided by the liv-
ing cells particularly UDP-rhamnose, which is not available commercial-
ly. Various types of living cells from seedlings to plant cell suspension
cultures have been employed as biocatalysts (Koen and Thiem, 1997).
9.3. GTs involved in hormonal regulation
The regulation of hormone level in plants is critical for plant growth
and adaptation to environmental changes. Glycosylation defines a key
mechanism in regulation of phytohormones in glycosidic form, with lit-
erature suggesting the presence of hormone glycosides except ethylene
(Fujioka and Yokota,2003; Woodward and Bartel, 2005; Ostrowski and
Jakubowska, 2014). The UDP-glycosyltransferases (UDP1 class) play an
important role in the biosynthesis of ether-type and ester-type of phy-
tohormone conjugates in plants. Widely known in plant kingdom, the
glycosylation of plant hormones defines an important regulatory mech-
anism for assessing the phytohormone levels in plants during growth
and developmental stages (Table 3). The phytohormones execute di-
verse functions like the storage and transport of hormones and their
degradation in metabolic pathway contrary to actingas signaling mole-
cules (Ostrowski and Jakubowska,2014). The glycosylation ofplant hor-
mones plays a critical role in influencing hormone homeostasis by
temporary inactivation and further by hydrolysis and conjugation with-
out de novo synthesis. The benefit of such system is to minimized energy
loss due to recycling of intact molecules and rapid responses (Jones and
Vogt, 2001).
The mechanism of hormone glycosylation is attributed to the chang-
es in substrate recognition by hormone or changes in their properties
(Kleczkowski and Schell, 1995). Studies involvingendogenous glycosyl-
ation of hormones have been reviewed, an example includes a recent
report that suggested the effect of O-glucosylation of cytokinin in
maize. Similar studies on glucosylation of zeatin, a naturally occurring
cytokinin identified a cis-zeatin O-glucosyltransferase (cisZOG1) from
730 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Z. mays and suggested that cis-zeatin and derivatives act as regulatory
elements in cytokinin homeostasis in plants (Martin et al., 2001a,
2001b). Another example includes the overexpression of zeatin O-
glucosyltransferase from Phaseolus lunatus L. in the roots and leaves of
the maize transgenic plant that led to higher fold expression and accu-
mulation of zeatin-O-glucoside and phenotype leading to growth retar-
dation and tassel seed formation characteristic of cytokinin-deficient
plants. These studies suggest that the regulation of cytokinin levels in
plant is influenced by O-glucosylation of cytokinins (Rodo et al., 2008).
Auxins constitute an important class of phytohormones, indole
acetic acid being a key example and exist in conjugated form. Glycosyl-
transferases catalyze the synthesis ofester conjugates through covalent
bond formation between the carboxyl group of the hormone and the
hydroxyl group present at the C1 position in β-D-glucopyranose hemi-
acetal form. In an attempt to address the function of IAA glycosides in
plants, several studies investigated the glucosylation of auxins and
their effect on the phytohormone metabolism. In a recent study by
Ostrowski and Jakubowska (2014), an IAA specific glucosyltransferase
was isolated from immature pea seeds and biochemically characterized.
The study revealed the possible role of the enzyme in the glycoprotein
modification in Pisum sativum. Interestingly, the glycosides of plant hor-
mones have been reported for almost all the classes of phytohormones
in plants. An abscisic acid glucosyltransferase was cloned and character-
ized from Phaseolus vulgaris L. (Palaniyandi et al., 2015). The identified
gene was able to glucosylate the abscisic acid into an inactive form
ABA-glucose ester,thereby playing a regulatory role in ABA homeostasis
during stress response and development in bean plant.
Further, several studies have demonstrated the role of glycosyltrans-
ferase in hormone glycosylation, like N-glycosylation of cytokinins
(Veach et al., 2003; Wang et al., 2011; Hou et al., 2014), auxins
(Szerszen et al., 1994; Jackson et al., 2001; Tognetti et al., 2010), salicylic
acid (Lee and Raskin, 1999; Song, 2006), brassinosteroids
(Poppenberger et al., 2005; Husar et al., 2011), and abscisic acid
(Priest et al., 2005; Palaniyandi et al., 2015; Liu et al., 2015). An
Table 2
Protein modeling and docking studies of plant glycosyltransferases.
Plant source Classification Template Resolution Substrates Catalytic
residues
Software used/type of
protein modeling
Reference
WsGTL1 and
WsGTL4 from
Withania
somnifera
GT-B type Chimeric
glucosyltransferase from
Actinoplanes
teichomyceticus,
3H4T
–β-sitosterol
Brassicasterol
Deactyl-16-DPA
Dehydro-epiandrosteron
Epoxypregnenolone
Ergosterol
Pregnenolone
Transandrosterone
Solasodine
Stigmasterol
24-methylene
cholesterol
Withaferin A
Withanolide A
Withanolide B
Withanolide D
Asp535
Phe506
Pro55
GENO3D Pandey et
al. (2015)
Flavonoid GT from
Withania
somnifera
GT-B type Medicago truncatula
UDP-glucuronosyl/
UDPglucosyltransferase,
2PQ6
(31% identity)
2.1 Å–3.1
Å
Diadzein
Apigenin
Luteolin
Naringenin
Genistein
Kaempferol
His18
Asp110
Trp352 Asn
353
Modeler 9v9,
homology modeling
Autodock vina 1.1.2.
Jadhav
et al.
(2012)
SlUGT5 from
Solanum
lycopersicum
UDP-glycosyltransferase
72 family
Arabidopsis UGT72B1
(60.5%
identity)
–Methyl salicylate
Guaiacol
Benzyl alcohol
Phenyl ethanol
Hydroquinone
Eugenol
His17 Glu
81 Phe 311
Modeler 9.7, homology
modeling,
AutoDock 4.2 tool
Louveau
et al.
(2011)
VvGT5 and VvGT6
from Vitis
vinifera
GT1 family VvGT1 –Kaempferol
UDP-glucose
Pro 19
Arg 140
Gln 373
Insight II modeling
homology modeling
Ono et al.
(2010a,
2010b)
CaUGT3 from
Catharanthus
roseus
GT-B type VvGT1 + kaempferol
complex,
(18% identity)
–Quercetin 3-O-glucoside His125 Asn
206
Molecular operating
environment software,
ASEDock
Masada
et al.
(2009)
BpUGT94B1 from
Bellis perennis
GT-B type MtUGT71G1(26%identity)
VvGT1(20% identity)
–Cyanidin 3ʹglucoside
6″-O-malonylglucoside
Delphinidin 3′O
glucoside
Arg (R25) Sybyl protein modeling,
Procheck,
homology modeling
Osmani
et al.
(2008)
UGT71G1 from
Medicago
truncatula
GT-B type A. orientalis GtfD (10%
identity)
2.6 Å Quercetin
Genistein
His22
Asp121
Multi-wavelength anomalous
dispersion (MAD) method,
RESOLVE, PROCHECK
Shao et al.
(2005)
UGT85B1 from
Sorghum bicolor
GT1 family GtfA and GtfB
(15% identity each)
–Mandelonitrile His23
Ser391
R201
Homology modeling program
COMPOSER, Sybyl software
(SYBYL)
Thorsoe
et al.
(2005)
UGT73A5 from
Dorotheanthus
bellidiformis
GT-B type UDP-glucosyltransferase
(Gtfb)
from A. orientalis, 1IIR
(14% identity)
–UDP-glucose
Betanidin
Glu378
His22
Homology modeling tool in
molecular operating
environment
(MOE)
AMBER
PROCHECK
PROSA II
Hans et al.
(2004)
731P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
Table 3
Glycosyltransferases involved in hormonal regulation in plants.
Glycosyltransferase Phytohormone Glycosylation product Reference
UGT71C5 from Arabidopsis thaliana Abscisic acid Abscisic acid-glucose ester Liu et al. (2015)
ABAGT from Phaseolus vulgaris L. Abscisic acid Abscisic acid-glucose ester Palaniyandi et al. (2015)
UGT73C14 from Gossypium hirsutum L. Abscisic acid ABA-glucoside Gilbert et al. (2013)
ABA GT from
Vigna angularis
Abscisic acid
2-trans-(+)-Abscisic acid
Trans-cinnamic acid
Abscisic acid-glucose ester Xu et al. (2002)
UGT71B6 from Arabidopsis thaliana Abscisic acid and structural analogs
PBI-413
PBI-410
PBI-82
Abscisic acid glucose ester Priest et al. (2005)
IAA GT from Pisum sativum Auxin
Indole-acetic-acid
1-O-IAA-glucose Ostrowski et al. (2015)
UGT84B1 from Arabidopsis thaliana Auxin
Indole-3-acetic acid
1-O-indole acetyl glucose ester Jackson et al., 2001
UGT74D1 from Arabidopsis thaliana Auxin
Indole-3-acetic acid
Indole-3-propionic acid
Indole-3-butyric acid naphthalene acetic acid
2,4-dichlorophenoxyacetic acid
Indole-3-carboxylic acid
Glucose esters Jin et al. (2013)
UGT73C6 from Arabidopsis thaliana Brassinosteroids
Brassinolide (BR)
Castasterone
Brassinolide-23-O-glucoside
BR malonyl glucosides
Castasterone -23-O-glucoside
Husar et al. (2011)
UGT73C5 from Arabidopsis thaliana Brassinosteroids
Brassinolide
Castasterone
Brassinolide-23-O-glucoside
Castasterone -23-O-glucoside
Poppenberger et al. (2005)
UGT85A1 from Arabidopsis thaliana Cytokinin
trans-zeatin
trans-Zeatin O-glucosides Jin et al. (2013)
UGT76C1 from Arabidopsis thaliana Cytokinin
Trans-zeatin
N6-isopentenyladenine
Cytokinin N-glucosides Wang et al. (2013)
UGT76C2 from Arabidopsis thaliana Cytokinin Cytokinin N-glucosides Wang et al. (2011)
UGT84B1 from Arabidopsis thaliana Indole-3-acetic acid Indole acetyl glutamate Jackson et al. (2002)
UGT74E2 from Arabidopsis thaliana Indole butryic acid IBA glucose Tognetti et al. (2010)
OsSGT from
Solanum tuberosum L.
Tuberonic acid
Salicylic acid
Tuberonic acid glucoside
Salicylic acid glucoside
Seto et al. (2009)
ZOG1 from
Phaseolus lunatus L.
Cytokinin
Zeatin
Zeatin-O-glucoside Rodo et al. (2008)
UGT74F1 and UGT74F2 from
Arabidopsis thaliana
Salicylic acid SA 2-O-beta-D-glucose
SA glucose ester
Dean and Delaney (2008)
InGTase1 from
Ipomoea nil
2-trans-abscisic acid
Indole-3-acetic acid
Salicylic acid
(±)-Jasmonic acid
Glucose esters Suzuki et al. (2007)
AtJGT1 from Arabidopsis thaliana Jasmonic acid
Dihydro jasmonic acid
Indole-3-acetic acid
Indole-3-propionic acid
Indole-3-butyric acid
Glucose esters Song et al. (2005)
ZOG1 from
Phaseolus lunatus
cisZOG1 from Zea mays
Cytokinin
Cis-zeatin
Trans-zeatin
Hydroxylated derivatives of benzyladenine (topolins)
O-glucoside of cis-zeatin
O-glucoside of trans-zeatin
O-glucoside of m-topolin
Mok et al. (2005)
UGT76C2 from Arabidopsis thaliana Cytokinin
N6-benzyladenine
Trans-zeatin
Cis-zeatin
Dihydrozeatin
N6-(+2-isopentenyl)-adenine
Kinetin
N6-benzyladenine-7-N-glucoside
N6-benzyladenine-9-N glucoside
Trans-zeatin-7-N-glucoside
Trans-zeatin-9-N-glucoside
Dihydrozeatin-7-N-glucoside
Dihydrozeatin-9-N-glucoside
N6-isopentenyladenine-7-N-glucoside
N6-isopentenyladenine-9-N-glucoside
Kinetin-3-N-glucoside
Hou et al. (2004)
cisZOG2 from
Zea mays
Cytokinin
Cis-zeatin
Cis-zeatin riboside
Cis-zeatin-O-glucoside
Veach et al. (2003)
cis-zeatin-O-GT from Zea mays Cytokinin
Zeatin
Cis-zeatin Martin et al. (2001b)
SA GTase from Nicotiana tabacum Salicylic acid SA 2-O-β-D-glucoside
Glucosyl salicylate
Lee and Raskin (1999)
AtSAGT1 from Arabidopsis thaliana Salicylic acid Glucosyl salicylic acid Song et al. (2009)
AtSGT1 from Arabidopsis thaliana Salicylic acid SA 2-O-β-D-glucoside
Salicylic acid glucose ester
Song (2006)
OsSGT1 from
Oryza sativa
Salicylic acid Salicylic acid O-beta-glucoside Umemura et al. (2009)
732 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
interesting example includes the isolation of a glucosyltransferase from
I. nil and its ability to glucosylate different phytohormones (Suzuki et al.,
2007). The enzyme exhibited a broad substrate preference ranging from
indole-3-acetic acid to salicylic acid and from 2-trans-abscisic acid to (±
)-jasmonic acid forming the respective glucose esters. Such studies
would shed light on the physiological roles of phytohormones GTs and
their regulatory mechanism in plants (Table 3). Moreover, it is assumed
that studies involving transgenics in sense and antisense orientation
would shed some light on the actual role of glycosylation mechanism
in regulation of hormone metabolism in plants.
9.4. GTs involved in modification of xenobiotics and detoxification of
pollutants
The glycosylation mechanism in plant secondary metabolism is an
efficient way to neutralize the toxic effect of foreign pathogens, pollut-
ants, toxic substances and xenobiotics. For example, the detoxification
of DON (trichothecene deoxynivalenol), a toxic substance produced by
Fusarium, a common pathogen of cereals such aswheat, maize and bar-
ley, is toxic to plant growth and development. A putative glycosyltrans-
ferase from Arabidopsis, UGT73C5, catalyzes the conversion of DON to
nontoxic DON 3-O-glucoside. Further, the overexpression of UGT73C5
in transgenic tobacco increased the resistance to DON (Poppenberger
et al., 2003) showing the detoxification role of GTs in plant.
Additionally, the activity of the enzymes against exogenous com-
pounds like insecticides, xenobiotics, pollutants and herbicides has
been reported (Lim et al., 2002; Loutre et al., 2003). Several Arabidopsis
UGTs are involved in the detoxification of 2, 4, 5-trichlorophenol (a xe-
nobiotic) (Messner et al., 2003) or the pollutant 3, 4-dichloroaniline
(DCA) (Loutre et al., 2003). Other examples include the detoxification
of 2, 4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-
7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by overexpression of GT
BX8 or GT BX 9 in transgenic Arabidopsis plants (von-Rad et al., 2001).
Ferreyra et al. (2013) identified and cloned a bifunctional C and O
glycosyltransferase involved in biosynthesis of insecticidal C-glycosyl
flavones, maysin and flavanone O-glycosides.
9.5. GTs involved in secondary metabolite biosynthesis
The role of glycosyltransferases and glycosylation mechanism in the
biosynthesis, storage and transport of secondary metabolites has been
demonstrated in several studies. In lignin biosynthesis, lignin mono-
mers (coumaryl, coniferyl and sinapyl alcohols) are required to be
translocated for polymerization to lignin. UGT72E2 and UGT72E3, the
recombinant glycosyltransferases of Arabidopsis, display 4-O-
glucosylation of phenylpropanoids (Lim et al., 2005b), suggesting that
these enzymes might play a role in the biosynthesis of lignin. The
exact mechanism of association between monolignol glycosylation
and lignin synthesis is not clear but it has been assumed that the down-
regulation of these GTs led the lowered glucoside levels of the mono-
mers of lignins in transformed Arabidopsis plants (Lanot et al., 2006).
Secondary metabolites such as monoterpenoids, hydroxybenzoic
acids and flavonols accumulate in plants both as aglycones and glycones.
Jones et al. (2003) isolated 3 GTs, UGT75C1, UGT78D2 and UGT79B1
from Arabidopsis involved in flavonol glycosides biosynthesis. Glycosyla-
tion is the terminal step in flavonol biosynthesis exhibiting a require-
ment of stability and translocation of molecule. Another example is the
biosynthesis of steviol glycosides in Stevia rebaudiana.Theplantextract
comprises of a mixture of as many as eight different glycosides of
diterpenoi steviol (the most intense sweet compound, 300 times sweeter
than sugar). The glycosylation mechanism begins with steviol and lead-
ing to formation of mono-, di-, tri- and tetraglycosides. S. rebaudiana
leaves comprise of stevioside and the tetraglycoside rebaudioside as
the major steviol glycosides (Wang and Hou, 2009). The first step
(glucosylation of the C-4 carboxyl position of steviolbioside) occurs in
plastids and the glycosides are transported into the leaf cells occupying
the vacuolar region. Functional genomic approaches identified three sa-
lient GTs shown to be involved in the synthesis of the major sweet
phyto-glucosides of S. rebaudiana. The heterologous expression and char-
acterization of the GTs revealed their regioselectivity towards steviol
glucosylating activities (Richman et al., 2005).
9.6. Stabilization of secondary metabolites
Glycosylation plays a key role in stabilization of phytomolecules. For
example, the anthocyanins which are the water soluble pigments pres-
ent in the vacuole constitute the secondary metabolites, widely taken
up for the studies related with glycoside formation versus stabilization.
Glycosylation together with the acylation of the sugars takes place in
the cytosol and then the conjugated anthocyanins are made transport-
able to get accumulated in the vacuoles. It is interesting to note the
mechanism of glycosylation may not participate in the color formation
of the anthocyanin structure but plays a critical role in the maintenance
of the structural integrity and stability of the flavylium cation. Further-
more, together with acylation process, complex anthocyanin structures
are formed(Strack and Wray, 1989). Furthermore, mutationalstudies in
grapevine suggested the absence of anthocyanidin glucosyltransferase
leading to anthocyanidin-less berries. Glycosylation at the C-3, C-5 or
C-7 position of anthocyanidins inhibits conversion into non-colored
forms, prevents its breakdown and improves stability of the molecule.
Therefore, the improved stability of glycosides limits their interaction
with the catabolic enzymes (Wajant et al., 1994). The cyanohydrins
(Moller and Seigler, 1998) and thihydroxymates (Halkier and Du,
1997) breakdowninto smaller constituents and glycosylation stabilizes
these molecules and protects the structure thereby facilitating their
accumulation in plant.
Another example of stabilization of molecule by glycosylation
process highlights the 2-O-glucosides of L-ascorbic acid, generally
more resistant to enzymatic and chemical oxidation compared to 6-O-
glucosides or aglycones (Yamamoto et al., 1990). The addition of sugar
moieties at specific position is essential in improving chemical stability
of the plant metabolites. The role of glycosylation mechanism in
stabilization of secondary metabolites is significant in case of cyanogen-
ic glycosides and glucosinolates. The addition of a sugar moiety to the
molecule prevents its degradation to aldehydes, cyanide or isothiocya-
nates and is essentialfor the maintenance of stability (Kahn et al., 1997).
9.7. Plant-microbe interactions
It is assumed that glycosylation mechanism might playrole in inter-
specific or intraspecific signaling in plant-microbe interactions. Bacteria
implicated in the plant to microbe interactions detect the presence of
plant secondary metabolites such as phenolics (Peters and Verma,
1990). It is believed that the glycosylation status of the phenolics may
be involved in influencing the signaling and perception response. In
Prunus avium, secondary metabolite phenolic glycosides enhance
phytotoxin synthesis in Pseudomonas, while the phenolic aglycones do
not exhibit inductive response indicating the involvement of glycosyla-
tion (Mo et al., 1995). Similar example shows that in symbiotic bacteria,
Rhizobium meliloti,luteolinactsasanod-gene inducer (Peters et al.,
1986) while luteolin 7-O-glucoside is a weak inducer and proceed via
free aglycone hydrolysis for signaling mechanism (Hartwig and
Phillips, 1991).
Several other studies indicate the role of glucosyltransferases in
response to pathogen infection. A studyby Jimenez et al. (2005) showed
that infection of Beta vulgaris with Pseudomonas syringae or
Agrobacterium tumefacians and mechanical wounding induced
glucosyltransferase (BvGT) expression in the plant. Furthermore, the
correlation between the BvGT antisense construct transient expression
and decrease in BvGT transcript accumulation suggested the role of GT
gene in betacyanin glucosylation and the reactive oxygen species
(ROS) produced by membrane associated NADPH oxidase may induce
733P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
BvGT expression. In a similar study, a UDP-glucosyltransferase induced
in response to P. syringae infection was cloned and characterized from
the bean plant. The cDNA clone was designated as Hra25 (for hypersen-
sitive reaction associated) and its activation in response to non-avr
(general signals) or avr-derived signals (specific) was studied in re-
sponse to P. syringae infection and wounding (Sullivan et al., 2001). An-
other study by Langlois-Meurinne et al. (2005) reported the function of
plant secondary metabolism glucosyltransferases in plant-pathogen in-
teractions. UGT73B3 and UGT73B5 from A. thaliana exhibited distinct
expression profiles on P. syringae infection, defense-signaling mutants
showing salicylic acid induced and methyljasmonate independent
expression. The study highlighted the contribution of UDP-GTs in
plant-microbe interactions and their role in hypersensitivity response.
The role of a glucosyltransferase gene during superoxide-dependent
cell death in A. thaliana was studied by Mazel and Levine in 2002.The
gene, UGT73B5 was induced by the superoxides, in the presence of
salicylic acid or cycloheximide and mechanical crushing and infection
with Botrytis cinerea. It was presumed that UGT73B5 may act in
transport of cellular components from the tissues undergoing cell
death, acting in coordination of metabolites during defensemechanisms
(Mazel and Levine, 2002). Furthermore, several studies have made
major contributions in deciphering the function of glucosyltransferase
class of enzymes in plant-microbe interactions namely conversion of
Deoxynivalenol (DON) produced by Fusarium graminearum to DON-3-
glucoside (Schweiger et al., 2013; Xin et al., 2014; Michlmayr et al.,
2015), induction of tobacco genes (TOGT) in response to fungal elicitors
and synthesis of conjugated aromatic metabolites (Fraissinet-Tachet
et al., 1998) and benzoxazinones synthesis in T. aestivum and Secale
cereale against microbial or herbivore attack (Sue et al., 2011). Role of
glucosyltransferase in Mi-mediated (nematode root-knot nematode
Meloidogyne species) nematode resistance was demonstrated (Schaff
et al., 2007). In the study, the root transcriptome of tomato nematode
resistant (Mi+) and susceptible (Mi−) cultivars of the plant, Motelle
and Moneymaker was studied following a time-course infection with
the pathogen. Furthermore, virus induced gene silencing of
glucosyltransferase reintroduced susceptibility to M. incognita in
Motelle plant highlighting the functional role of the gene in resistance
to the nematode (Schaff et al., 2007).
At the intraspecies levels, glycosylation process may also influence
plant-to-plant signaling related responses. An example is flavonol
aglycones, kaempferol and quercetin in maize and petunia, which are
involved in the germination of pollen while the corresponding
glycosides are shown to be non-responsive (Mo et al., 1992).
9.8. Metabolic engineering of crops
The genetic manipulation of economically viable crops is an impor-
tant biotechnological application for improving food (nutritional con-
tent) or the quality of crop. By employing the technique of enzyme
immobilization, a recombinantly cloned GT from pummelo converted
the bitter limonoids of lemon juice into tasteless glycosides (Karim
and Hashinaga, 2002). The techniques involving isolation and establish-
ment of enzymatic activities and biological roles of GTs in plants would
serve as a platform for crop improvement. GTs play diverse functional
roles in plant secondary metabolism from defense responses to detoxi-
fication and regulation of hormonelevels to increase in stability of mol-
ecules and would be an ideal candidate for creating transgenics with
improved traits. For instance, GTs involved in homeostasis of plant hor-
mones such as auxins, cytokinins and brassicasteroids offers new pros-
pects in creating crops with desired phenotypic traits by manipulating
the hormone levels which would further influence the growth and
development of the plants. Other desired features of crop engineering
include insertion of GTs involved in detoxification of pesticides and
xenobiotics to enhance food quality and safety, to create plants with
increased resistance to abiotic and biotic stress and increased levels of
glycosides in food with antioxidant and anticancer properties.
Recently, a rice glycosyltransferase phylogenomic database was cre-
ated by Cao et al. (2008),whoidentified rice-diverged glycosyltransfer-
ases. It would provide promising lead in studying GTs in rice and other
crop plants. Strategies involving manipulation of active site residues
through docking studies and generation of mutants through knockouts
and gene silencing are the emerging research trends in genetic engi-
neering of crops and would establish the biological role of GTs in plants.
9.9. Pharmacological studies using UGTs
Many of the phytomolecules are known to exist as glycosides namely
flavonoids, hormones, sweeteners, antibiotics, and alkaloids (Blanchard
and Thorson, 2006).Thepresenceofcarbohydratemoietyisacriticalre-
quirement for pharmacological parameters of drug and its activity (Kren
and Martínkova, 2001). Techniques in molecular biology and biochemis-
try have made significant contributions in biosynthetic studies of glyco-
sylated molecules both at genomics and metabolomics level. It has
been reported that the attachment of a sugar moiety to a molecule en-
hances its solubility and hence, bioavailability (Thorson et al., 2001).
Studies have reported that glycosylation of bioactive compounds at spe-
cific positions modify their pharmaceutical properties (Kren and
Martínkova, 2001; Mijatovic et al., 2007). However, synthesis of specific
glycoconjugates employing organic chemistry is difficult and enzymatic
glycosylation is a better alternative strategy for biosynthesis of desired
glycoconjugates. These parameters are important aspects to be consid-
ered in rational drug designing. The availability of plethora of bioactive
natural products with medicinal properties and their glycosylation
mechanism provides a key platform for identification of drug targets
and pharmacophore development. Studies involving active site muta-
tions would probably confer new property/function to the existing en-
zyme with an aim of refinement in certain specifictraits.
The use of GT enzymes in pharmaceutical industry holds good pros-
pects but the unavailability of nucleotide activated sugars has posed a
limitation on further applications of these enzymes (Luzhetskyy et al.,
2007). Another aspect in drug engineering is the availability of informa-
tion of a vast set of GT genes which may catalyze side chains formation
and glycosylate molecules of diverse properties. With the progress in
whole-genome sequencing projects, novel GTs are being identified
and their pharmacological prospects are explored immensely.
10. Future prospects in glycosyltransferase research
The future prospects of plant secondary metabolic glycosyltransfer-
ases in commercial and economic applications look promising. With
the range of activities catalyzed by GTs from glycoconjugate synthesis
to detoxification of pollutants and xenobiotics to drug targeting and
crop engineering, the benefits of glycosylation are immense. The rising
trends in world population are creating more consumption demands
and genetic engineering of economically viable crops for quality im-
provement is a step ahead. Mutational studies in plants including
sense and antisense approaches would demonstrate the biological role
of GTs in plants. For instance, GTs involved in homeostasis of plant hor-
mones such as cytokinins, brassicasteroids and auxins hold good pros-
pects in creating crops with desired phenotypic traits by manipulating
the hormone levels which would further influence the growth and de-
velopment of the plants. Other desired features of crop engineering in-
clude insertion of GTs involved in detoxification of pesticides and
xenobiotics to enhance food quality and safety, to create plants with in-
creased resistanceto abiotic and biotic stress and increased levels of gly-
cosides in food with antioxidant and anticancer properties.
Further, it has been demonstrated that addition of a carbohydrate
moiety to a metabolite leads to enhanced solubility and reduced toxicity
thereby improvement in bioavailability. These properties are important
criteriafor drug designing. The glycosylated molecules serve as an ideal
target in pharmacophore development. Certain GTs with broad
spectrum activityare permissible candidates for enzyme manipulations
734 P. Tiwari et al. / Biotechnology Advances 34 (2016) 714–739
with some others with stringent specificity which are disadvantageous
and a hindrance in glycoengineering. However, theactive site mutation-
al studies have been a remarkable achievement in creating novel
“chimeras”with the desired traits and properties.
The biotechnological applications of GTs make them superior candi-
dates in several important areas of research. However, certain aspects
on GT studies still need to be explored in detail. The availability of crys-
tal structures of very few plant GTs (triterpene GT from M. truncatula,
flavonoid GT from Avena sativa and few more) is a major limitation in
GT research. The correlation between three dimensional structure and
functional properties of the enzyme (acceptor binding specificity) is
awaited. The elucidation of GT crystal structure would shed light on
the functional role of GTs and the biosynthesis of phytoconstituents
with pharmacological significance in important medicinal plants.
The evolution of catalytic preferences for substrate highlights
regioselectivity of GTs for O glycosylation as compared to relatively
very few C, N and S glycosylation respectively. Studies by Lim et al.
(2002) and Loutre et al. (2003) demonstrated that a bifunctional GT
from Arabidopsis GT72B1 catalyzes an O-glycosidic linkage with 3,
4-dihydroxybenzoic acid and an N-glucosidic bond with 3, 4-
dichloroaniline (a pollutant). GTs display enzymatic catalysis for
oxygen, carbon, nitrogen and sulfur and mostly involved in heteroatom
glycosylation (O-glycosylation, N-glycosylation and S-glycosylation)
except C-glycosylation GTs (Chang et al., 2011). Recently, a bifunctional
maize GT was identified displaying C-glycosylation for natural insecti-
cide, maysin as well as O-glycosylation for flavones as substrates. The
evolution of dual catalytic mechanism in the maize GT highlights the
multiple functional roles, one in defense mechanism against insects
while a second as a detoxification mechanism for flavanone
O-glycosides and their increased stability (Ferreyra et al., 2013). Such
dual catalytic role played by GTs forms a prospective strategy for the ge-
netic engineering of the candidate genes in introduction/improvement
of the desired features in recombinant GTs. The presence of bifunctional
GTs in nature suggests the evolution of a precise mechanism of enzy-
matic catalysis, however identification of very few (UGT708A6 from
Z. mays and A. thaliana GT72B1) is an area of consideration and investi-
gations. The predominance of O glycosylation in GTs defines a con-
served mechanism of catalysis showing a remarkable preference for
OH groups as sugar acceptors. Considering the diverse applications
and pharmacological significance of the GT superfamily in plant second-
ary metabolism, the isolation and genetic manipulation studies form an
interesting and prospective field in drug designing studies.
Leloir (nucleotide-dependent) GT enzymes are potential candidates
as biopharmaceuticals, in development of vaccines and small molecule
medicines. GT engineering (involving sequence analysis or structural
manipulations) includes the creation of chimeric GTs created by
mutational studies. Successful attempts include flavonoid GT from
A. thaliana, bacterial aminoglycoside, glycopeptide, and angucycline
GTs (Brazier et al., 2007; Hansen et al., 2009a, 2009b; Hoffmeister
et al., 2001, 2002; Krauth et al., 2009; Park et al., 2009; Truman et al.,
2009). Further, glycoengineering leading to modifications in either
donor or acceptor binding sites may also improve thecatalytic efficiency
of the enzyme, e.g. includes the positional mutations in M. truncatula
UGT85H2, (GT-B fold) where mutations were introduced both in
donor and acceptor sites, and lead to a substantial as high as 54-fold
enhancements in thecatalysis (Modolo et al., 2009). Therefore, research
on glycosyltransferases particularly those on plant GTs is gaining
momentum and GT enzymes exhibit good prospects as novel targets
in drug designing and development.
Acknowledgments
Authors are thankful to Council of Scientific and Industrial Research
(CSIR), New Delhi for the financial grant to conduct research on
glycosyltransferases in the networking project, NWP-09. The authors ac-
knowledge the director of Central Institute of Medicinal and Aromatic
Plants (CSIR-CIMAP) for the constant encouragement and support. PT
is thankful to CSIR, New Delhi for the award of Senior Research Fellow-
ship. RSS and NSS are responsible for the study concept, design, analysis
and interpretation of the article. PT contributed in literature collection
and writing of the manuscript. RSS and NSS have critically read the man-
uscript. NSS holds the decision to submit the review in Biotech Advances.
Finally, we would like to extend our sincere thanks to everyone whose
support directly or indirectly, have made this manuscript possible.
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