Fig 2 - uploaded by Maizom Hassan
Content may be subject to copyright.
Identification of Geranial as Reaction Product Following Incubation of P. minus Cell-Free Extract with Geraniol. A, Separation of authentic geraniol, geranial, and neral by GCMS. Peak 1, neral; peak 2, geraniol; peak 3, geranial. Retention times of 16.41 min, 16.84, and 17.42 min were obtained for neral, geraniol, and geranial respectively. B, GC-MS analysis following incubation of cell-free extract with geraniol and NADP þ. A new peak (peak 3, geranial) with a retention time of 17.41 min was seen. C, Peak 3 (geranial) was absent from control incubation without cell-free extract.
Source publication
NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (p...
Contexts in source publication
Context 1
... determine whether geranial was a product of the geraniol-DH reaction, GC-MS analysis was performed. A 1:1 mixture of authentic geraniol and citral is shown in Fig. 2. Citral is a mixture of geranial (trans) and neral (cis) in an approximately 3:2 ratio. 4) The retention times of the three resulting peaks for neral (cis-isomer), geraniol, and geranial (trans-isomer) were 16.41 min, 16.84 min, and 17.42 min respectively (Fig. 2A). The enzyme reaction assay containing NADP þ and geraniol was incubated ...
Context 2
... analysis was performed. A 1:1 mixture of authentic geraniol and citral is shown in Fig. 2. Citral is a mixture of geranial (trans) and neral (cis) in an approximately 3:2 ratio. 4) The retention times of the three resulting peaks for neral (cis-isomer), geraniol, and geranial (trans-isomer) were 16.41 min, 16.84 min, and 17.42 min respectively (Fig. 2A). The enzyme reaction assay containing NADP þ and geraniol was incubated in the presence and the absence of a cell- free extract of P. minus leaves at 37 C for 3 h and then analyzed by GC-MS. As shown in Fig. 2B, a new peak with a retention time of 17.41 min, similar to the retention time of authentic geranial, appeared when the ...
Context 3
... resulting peaks for neral (cis-isomer), geraniol, and geranial (trans-isomer) were 16.41 min, 16.84 min, and 17.42 min respectively (Fig. 2A). The enzyme reaction assay containing NADP þ and geraniol was incubated in the presence and the absence of a cell- free extract of P. minus leaves at 37 C for 3 h and then analyzed by GC-MS. As shown in Fig. 2B, a new peak with a retention time of 17.41 min, similar to the retention time of authentic geranial, appeared when the reaction mixture was incubated with the cell-free extract, geraniol, and NADP þ . This peak was not detected in the control reaction, without the cell-free extract (Fig. 2C), and it was also absent from the control ...
Context 4
... at 37 C for 3 h and then analyzed by GC-MS. As shown in Fig. 2B, a new peak with a retention time of 17.41 min, similar to the retention time of authentic geranial, appeared when the reaction mixture was incubated with the cell-free extract, geraniol, and NADP þ . This peak was not detected in the control reaction, without the cell-free extract (Fig. 2C), and it was also absent from the control with cell-free extract but without geraniol (data not shown). These results confirmed that geranial was the enzymatic product, and that geraniol-DH was the enzyme catalyzing the oxidation reaction of geraniol in P. ...
Similar publications
NADP þ-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glyc-erol, 2-mercaptoethanol, thiourea, and phenylmethyl-sulfonylfluoride in tricine-NaOH buffer (...
Citations
... Based on previous studies of alcohol dehydrogenase, several inhibitors and metal ions were selected (Inoue, Tsuji and Uritani 1984;Singh Sangwan, Singh-Sangwan and Luthra 1993;Hallahan et al. 1995;Hassan et al. 2012;Ahmad-Sohdi et al. 2015) and standardized to 2.0 mm concentration. The effects of inhibitors and metal ions on enzyme activity were studied by first incubating this purified PxFoLDH (12.5 μg) for 10 min at 55°C. ...
... Based on these findings, PxFoLDH functions optimally in alkaline conditions. A similar result of optimal pH value was reported in farnesol dehydrogenases from A. aegypti, P. minus, and I. batatas (Inoue, Tsuji and Uritani 1984;Mayoral et al. 2009a;Ahmad-Sohdi et al. 2015), insect alcohol dehydrogenases (Gasperi et al. 1994;Noge et al. 2008;Wang et al. 2011), and to the plant terpene alcohol dehydrogenases (Potty and Bruemmer 1970;Ikeda et al. 1991;Hallahan et al. 1995;Iijima et al. 2006;Hassan et al. 2012). ...
... Chelating agent for the Fe ion, 2,2 -dipyridil, and sodium azide caused approximately 20%-50% inhibition in PxFoLDH activity. The activity of P. minus and I. batatas farnesol dehydrogenases (Inoue, Tsuji and Uritani 1984;Ahmad-Sohdi et al. 2015), and other terpene alcohol dehydrogenases (Potty and Bruemmer 1970;Kjonaas, Venkatachalam and Croteau 1985;Ikeda et al. 1991;Singh Sangwan, Singh-Sangwan and Luthra 1993;Hallahan et al. 1995;Croteau, Lee Hooper and Felton 2004;Hassan et al. 2012) were also greatly inhibited by sulfhydryl agents. The ability of sulfhydryl agents to inactivate PxFoLDH suggests that the active site of this enzyme consists of a sulfhydryl group that is essential for its catalytic activity. ...
Juvenile hormone III (JH III) plays an important role in insect reproduction, development, and behavior. The second branch of JH III production includes oxidation of farnesol to farnesal by farnesol dehydrogenase. This study reported the identification and characterization of Plutella xylostella farnesol dehydrogenase (PxFoLDH). Our results showed that PxFoLDH belongs to the short-chain dehydrogenase/reductase superfamily, consisting of a single domain with a structurally conserved Rossman fold, an NAD(P) (H)-binding region and a structurally diverse C- terminal region. The purified enzyme displayed maximum activity at 55 °C with pH 9.5 and was stable in the temperature below 70 °C. PxFoLDH was determined to be a monomer with a relative molecular weight of 27 kDa and highly specific for trans, trans-farnesol and NADP+. Among analog inhibitors tested, farnesyl acetate was the most effective inhibitor with the lowest Ki value of 0.02 µM. Our findings showed this purified enzyme may represent as NADP+-farnesol dehydrogenase.
... One the other hand, potential GES of citronellol may decide on the citronellol accumulation on to a greater extent by inducing citronellol synthesis. Whereas, the studies of dehydrogenases of geraniol or nerol are greatly about geraniol acid and haven't identified the one for citronellol transformation until now (Hassan et al., 2012;Tan et al., 2019a;Tan et al., 2019b), much less to the indistinguishable identification of geraniol or citronellol GES which is hidden in ambiguous function of terpenoid synthase family (Magnard et al., 2015). We prefer that the high citronellol accumulation more result from the induced response of an assumed GES. ...
Rosa rugosa is an important natural perfume plant in China. Rose essential oil is known as ‘liquid gold’ and has high economic and health values. Monoterpenes are the main fragrant components of R. rugosa flower and essential oil. In this study, a member of the hydrolase gene family RrNUDX1 was cloned from Chinese traditional R. rugosa ‘Tang Hong’. Combined analysis of RrNUDX1 gene expression and the aroma components in different development stages and different parts of flower organ, we found that the main aroma component content was consistent with the gene expression pattern. The RrNUDX1 overexpressed Petunia hybrida was acquired via Agrobacterium -mediated genetic transformation systems. The blades of the transgenic petunias became wider and its growth vigor became strong with stronger fragrance. Gas chromatography with mass spectrometry analysis showed that the contents of the main aroma components of the transgenic petunias including methyl benzoate significantly increased. These findings indicate that the RrNUDX1 gene plays a role in enhancing the fragrance of petunia flowers, and they could lay an important foundation for the homeotic transformation of RrNUDX1 in R. rugosa for cultivating new R. rugosa varieties of high-yield and -quality essential oil.
... Many oxidoreductases have been reported to act on alcohol groups of donor substrates. These include geraniol dehydrogenases, which require NADP þ /NAD þ as cofactors (Hassan et al. 2012), and cinnamyl alcohol dehydrogenases grouped in flavoproteins (Lauvergeat et al. 1995). The oxidoreductase in L. erythrorhizon cells catalyzing (Z)-3 00 -OH-GHQ dehydrogenation requires NADP þ as its sole cofactor but could not use NAD þ (Fig. 5). ...
... These results suggested that the enzyme that oxidizes (Z)-3 00 -OH-GHQ in L. erythrorhizon cells is an ADH-type oxidoreductase. This dehydrogenase had an optimum pH of about 8.5 ( Supplementary Fig. S2) and K m values of 19.89 ± 1.27 and 5.08 ± 0.19 lM for (Z)-3 00 -OH-GHQ and NADP þ , respectively, comparable to or lower than those of other plant ADHs (Hallahan et al. 1995, Hassan et al. 2012. ...
Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3"-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3"-alcoholic group of (Z)-3"-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3"-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although non-enzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin producing and non-producing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.
... It is possible, therefore, that HcADH2 and HcADH3 also have a defensive function in H. coronarium vegetative organs. Hassan et al. (2012) reported that DH I and II Polygonum minus geraniol dehydrogenase can catalyse the oxidation of geraniol to geranial, and they might be involved in monoterpene alcohol metabolism and also have their high specificity for allylic alcohols. The recombinant HcADH3 enzyme can use monoterpenoids geraniol as a catalytic substrate. ...
In this study, the full cDNA sequences of HcADH2 and HcADH3 were cloned from Hedychium coronarium . The amino acid sequences encoded by them contained three most conserved motifs of short-chain alcohol dehydrogenase (ADH), namely NAD ⁺ binding domain, TGxxx[AG]xG and active site YxxxK. The highest similarity between two genes and ADH from other plants was 70%. Phylogenetic analysis showed that they belonged to a member of the short-chain dehydrogenases/reductases 110C subfamily, but they were distinctly clustered in different clades. Real-time polymerase chain reaction analyses showed that HcADH2 was specifically expressed in bract, and it was expressed higher in no-scented Hedychium forrestii than other Hedychium species, but was undetectable in Hedychium coccineum . HcADH3 was expressed higher in the lateral petal of the flower than in other vegetative organs, and it was expressed the most in H. coronarium that is the most scented among Hedychium species, and its expression levels peaked at the half opening stage. HcADH2 and HcADH3 had almost no significant expression in leaves, but HcADH2 was expressed in response to external stimuli. The mechanical injury and methyl jasmonate (MeJA) treatment could induce expression of HcADH2 in leaves, whereas HcADH3 could have an induced expression only by MeJA. The recombinant HcADH3 protein, but not HcADH2, expressed in Escherichia coli -catalysed conversion of geraniol into citral. It was speculated that HcADH3 had an induced expression in vegetative organ of H. coronarium and took part in monoterpenoid biosynthesis in H. coronarium flowers, but the role of HcADH2 is relevant only for defensive reactions.
... Biosynthesis of citral has been well elucidated, which occurs in the first oxidative step of geraniol degradation pathway (Kanehisa and Goto 2000;Noge et al. 2008) (Figure 1). Early research on the pathway was mainly based on the bioconversion products of microorganisms (Cantwell et al. 1978), while later attempts were focused on the characterization of the substrate specificity and kinetic properties of the natively purified and recombinant enzyme involved in citral production (Ikeda et al. 1991;Wolken and van der Werf 2001;Iijima et al. 2006;Noge et al. 2008;Hassan et al. 2012;Luddeke et al. 2012). Throughout the last decade, geraniol dehydrogenase (GeDH, EC 1.1.1.183) ...
... The specific nerol-oxidizing activity of PmNeDH as a result of its highest affinity towards nerol was different from the previously reported P. minor GeDH isoenzymes (PmGeDH), which have the highest preference to use geraniol as substrate and produce geranial as its product (Hassan et al. 2012). Due to their highly related role in P. minor, PmNeDH can be regarded as a paralogous protein of PmGeDHs that probably arose from a gene duplication event but eventually received a new function that was specific to nerol. ...
Citral is a mixture of neral and geranial, which are of great interest to the fragrance industry due to its lemon-scented aroma. A newly characterized nerol dehydrogenase of Persicaria minor (PmNeDH) from our recent findings has shown a capacity to convert citral from nerol. Differential gene expression analysis revealed that the expression level of PmNeDH was highly upregulated during early treatment of several stress-related phytohormones i.e. methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA). SA and ABA were shown to have a prolonged effect on PmNeDH expression level until second day of treatment. The findings were in agreement with the cis-regulatory elements predicted from the gene promoter. The phylogenetic relationship of PmNeDH with its homologs from the medium-chain dehydrogenases/reductases (MDR) superfamily was also mentioned. In this study, we proposed a possible biological function of PmNeDH gene in P. minor, which might play significant roles in plant defence mechanism.
... Compared to the full range of terpene secondary modifications known so far, only a few genes encoding enzymes involved in these reactions have been characterised. For example, oxidation of geraniol in geranial is mediated by an alcohol dehydrogenase (ADH) in basil (Iijima et al., 2006), ginger (Iijima et al., 2014), perilla (Sato-Masumoto and Ito, 2014), the orchid Caladenia (Xu et al., 2017) and Polygonum (Hassan et al., 2012). Gene encoding for the enzyme mediating acetylation of geraniol and citronellol has only been characterised once FIGURE 1 | Glandular trichome and mono and sesquiterpenes biosynthetic pathways in Pelargonium. ...
Pelargonium genus contains about 280 species among which at least 30 species are odorant. Aromas produced by scented species are remarkably diverse such as rose, mint, lemon, nutmeg, ginger and many others scents. Amongst odorant species, rose-scented pelargoniums, also named pelargonium rosat, are the most famous hybrids for their production of essential oil (EO), widely used by perfume and cosmetic industries. Although EO composition has been extensively studied, the underlying biosynthetic pathways and their regulation, most notably of terpenes, are largely unknown. To gain a better understanding of the terpene metabolic pathways in pelargonium rosat, we generated a transcriptome dataset of pelargonium leaf and used a candidate gene approach to functionally characterise four terpene synthases (TPSs), including a geraniol synthase, a key enzyme responsible for the biosynthesis of the main rose-scented terpenes. We also report for the first time the characterisation of a novel sesquiterpene synthase catalysing the biosynthesis of 10-epi-γ-eudesmol. We found a strong correlation between expression of the four genes encoding the respective TPSs and accumulation of the corresponding products in several pelargonium cultivars and species. Finally, using publically available RNA-Seq data and de novo transcriptome assemblies, we inferred a maximum likelihood phylogeny from 270 pelargonium TPSs, including the four newly discovered enzymes, providing clues about TPS evolution in the Pelargonium genus. Notably, we show that, by contrast to other TPSs, geraniol synthases from the TPS-g subfamily conserved their molecular function throughout evolution.
... Geraniol dehydrogenase (GeDH, EC 1.1.1.183) is thus far the only enzyme that has been assigned to the production of citral through oxidation of nerol and geraniol [5,[9][10][11][12][13]. In plants, the production of geraniol from its precursor geranyl pyrophosphate (GPP) and its oxidation to geranial have been well established [14]. ...
... P. minor is a perennial aromatic herb that belongs to the Polygonaceae family, which is known for its high levels of aliphatic aldehyde (76.59 %) in essential oil [17]. Previously, two GeDH isoenzymes were successfully purified and characterised from P. minor, where the purified enzymes are able to catalyse the oxidation of geraniol, nerol and several allylic alcohols [11]. Although biosynthesis of citral have been reported in many species, it is commonly regarded that citral is synthesised from geraniol and nerol by GeDH [9,[11][12][13]. ...
... Previously, two GeDH isoenzymes were successfully purified and characterised from P. minor, where the purified enzymes are able to catalyse the oxidation of geraniol, nerol and several allylic alcohols [11]. Although biosynthesis of citral have been reported in many species, it is commonly regarded that citral is synthesised from geraniol and nerol by GeDH [9,[11][12][13]. ...
Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.
... Geraniol is then synthesized by geraniol synthase (Iijima et al. 2004) and oxidized by geraniol dehydrogenase (EC 1.1.1.183) into geranial (Hassan et al. 2012). Previous study by Iijima et al. (2006) indicated that geraniol dehydrogenase is capable of oxidizing geraniol into citral (a mixture of geranial and neral; isomeric monoterpene aldehyde; Iijima et al. 2004). ...
... Previously, geraniol and geranial have been discovered in the study of Polygonum sp. (Persicaria minor) essential oils (Baharum et al. 2010;Hassan et al. 2012) where geraniol dehydrogenase has been successfully purified and characterized (Hassan et al. 2012). Moreover, citral dehydrogenase activity was also identified in crude extract of P. minor (this study). ...
... Previously, geraniol and geranial have been discovered in the study of Polygonum sp. (Persicaria minor) essential oils (Baharum et al. 2010;Hassan et al. 2012) where geraniol dehydrogenase has been successfully purified and characterized (Hassan et al. 2012). Moreover, citral dehydrogenase activity was also identified in crude extract of P. minor (this study). ...
Plants emit semiochemicals as alarm signals upon attack by herbivores or insect pests. Complex insect-plant interaction through alarm pheromones can be manipulated to improve crop protection. Geraniol, citral and geranic acid are monoterpenoid compounds from plants and they play a role as semiochemical alarm pheromones. In plants, the oxidation of geraniol into geranic acid is catalyzed by two oxidoreductases, geraniol dehydrogenase and citral dehydrogenase. In this study, citral dehydrogenase isoenzymes from Persicaria minor (Polygonum minus) leaves were purified to homogeneity and characterized. Enzyme purification through Toyopearl GigaCap Q-650 M column chromatography at pH 7.5 produced two activity peaks, suggesting the existence of two citral dehydrogenase isoenzymes. Both isoenzymes were different in isoelectric point and kinetic parameters but similar in pH and optimal temperature as well as in substrate specificity. Findings from this study will provide a basic understanding for the development of recombinant production of these particular enzymes. Further studies on molecular structure involved could be exploited in transgenic plant as an integrated pest management strategy.
Keywords
Persicaria minor (Polygonum minus Huds.) Citral dehydrogenase Isoenzymes Alarm pheromone Geranic acid
... Previous chemical studies of P. minor have shown that P. minor essential oil contains mainly aldehydes and terpenes (Baharum et al., 2010;Ahmad et al., 2014), and sesquiterpenes are found predominantly in the flower (Prota et al., 2014). A few enzymes involved in flavonoid and terpenoid metabolite biosynthesis including geraniol dehydrogenase, chalcone synthase, and farnesol dehydrogenase have been identified in P. minor (Hassan et al., 2012;Roslan et al., 2012;Ahmad Sohdi et al., 2015). Recently, a putative P. minor sesquiterpene synthase (PmSTS) gene (GenBank: JX025008) has been isolated. ...
Background
Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment.
Methods
The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server.
Results
Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases.
Discussion
The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.
... Pathway B included three reaction steps: 1) geraniol dehydrogenation to give citral; 2) double bond reduction of citral to form citronellal; 3) a final reducing step to produce citronellol. Geraniol dehydrogenase, which catalyzes the first step in pathway B, has been identified in several plant species including P. graveolens and Z. officinale [10,[18][19][20][21][22]. However, enzymes physiologically catalyzing the latter two steps of pathway B have not yet been identified. ...
Pelargonium graveolens L'Her, also referred to as rose geranium, is a popular herbal plant with typical rosy fragrance largely based on the blend of monoterpenoid constituents. Among them, citronellol, which is biosynthesized from geraniol via double bond reduction, is the most abundant scent compound. In this study, three 12-oxophytodienoic acid reductases (PgOPR1-3) have been cloned from P. graveolens, as possible candidates for the double-bond reductase involved in citronellol biosynthesis. The bacterially expressed recombinant PgOPRs did not reduce geraniol to citronellol, but stereoselectively converted citral into (S)-citronellal in the presence of NADPH. Thus, the alpha,beta-unsaturated carbonyl moiety in the substrate is essential for the catalytic activity of PgOPRs, as reported for OPRs from other plants and structurally related yeast old yellow enzymes. PgOPRs promiscuously accepted linear and cyclic alpha,beta-unsaturated carbonyl substrates, including methacrolein, a typical reactive carbonyl compound. The possible biotechnological applications for PgOPRs in plant metabolic engineering, based on their catalytic properties, are discussed herein.
