Vitamin K1 (phylloquinone): function, enzymes and genes

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From: Chloë van Oostende, Joshua R. Widhalm, Fabienne Furt, Anne-Lise Ducluzeau
and Gilles J. Basset, Vitamin K1 (Phylloquinone): Function, Enzymes and Genes.
In Fabrice Rébeillé and Roland Douce, editors:
Advances in Botanical Research, Vol. 59,
Amsterdam, The Netherlands, 2011, pp. 229-261.
ISBN: 978-0-12-385853-5
© Copyright 2011 Elsevier Ltd.
Academic Press.

Vitamin K
1
(Phylloquinone): Function, Enzymes and Genes
CHLOE
¨VAN OOSTENDE, JOSHUA R. WIDHALM,
FABIENNE FURT, ANNE-LISE DUCLUZEAU
AND GILLES J. BASSET
1
Center for Plant Science Innovation, University of Nebraska-Lincoln,
Lincoln, Nebraska, USA
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
II. Structure and Chemistry of Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
III. Biochemical Roles of Vitamin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
A. Vertebrates... . . . . . . ...... . . . . . . ....... . . . . . . ...... . . . . . ........ . . . . . ...... . . . . . 233
B. Plants and Cyanobacteria .. .. .. .. ... .. .. .. .... .. .. .. ... .. .. .. .... .. .. .. ... .. 236
IV. Detection and Distribution of Phylloquinone in Plants . . . . . . . . . . . . . . . . . . 238
A. Detection....... . . . . . . ...... . . . . . . ....... . . . . . . ...... . . . . . ........ . . . . . ...... . . . 238
B. Tissular Distribution . .. . .. .. .. .. .. . .. .. .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. . .. .. .. . 238
C. Subcellular Distribution . .. ..... .. .. .... .. .. .... .. .. ... .. .. .... .. .. .... .. .. .. 238
V. Phylloquinone Biosynthesis in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
A. Early Work . .. ..... .. .. .. ... .. .. .... .. .. ...... .. .. .... .. .. ..... .. .. ..... .. .. ... 240
B. Isochorismate Synthase/PHYLLO (Reactions 1–4) ....... . . . . . ....... . . 242
C. OSB-CoA Ligase (Reaction 5) . . . . . . ...... . . . . . . ....... . . . . . . ...... . . . . . ... 244
D. DHNA-CoA Synthase/DHNA-CoA Thioesterase (Reactions 6/7)... 244
E. DHNA Phytyl Transferase (Reaction 8)... . . . . . ........ . . . . . ...... . . . . . . . 246
F. Demethylphylloquinone Methyltransferase (Reaction 9) .. . . . . . ...... . 246
G. Mutant Phenotype.... . . . . . ....... . . . . . . ....... . . . . . ....... . . . . . ....... . . . . . . . 247
H. Subcellular Localization of Phylloquinone Biosynthetic Enzymes ... 247
VI. Evolution of Naphthoquinone Biosynthesis in Photosynthetic
Eukaryotes ................................................................ 250
VII. Phylloquinone Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
1
Corresponding author: E-mail: gbasset2@unl.edu
Advances in Botanical Research, Vol. 59 0065-2296/11 $35.00
Copyright 2011, Elsevier Ltd. All rights reserved. DOI: 10.1016/B978-0-12-385853-5.00001-5
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VIII. Engineering of Phylloquinone in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
IX. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
ABSTRACT
Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone) is a conjugated isoprenoid
that serves as a cardinal redox cofactor in plants and some cyanobacteria. In humans
and other mammals, it is required as a vitamin (vitamin K
1
) for blood coagulation
and bone metabolism. Until recently, the biosynthesis of phylloquinone in plants was
considered identical to that of menaquinone (vitamin K
2
) in facultative anaerobic
bacteria. It resulted that most of the plant research on phylloquinone focused histori-
cally on the study of its function, while very little was done on its metabolism per se.
There is today, tough, compelling evidence that plants have evolved an unprecedented
metabolic architecture to synthesize phylloquinone, including extraordinary events of
gene fusion, highly divergent enzymes and a separated compartmentalization in
chloroplasts and peroxisomes. Phylogenetic reconstructions also demonstrate that
the plant genes involved in the formation of phylloquinone display a high degree of
evolutionary chimerism owing to multiple events of horizontal gene transfer and gene
losses. Plant phylloquinone biosynthesis is also connected via shared intermediates to
the metabolism of salicylate, tocopherols, chlorophylls, and in some species to
anthraquinones.
I. INTRODUCTION
The discovery of vitamin K arose from the observation in the late 1920s and
early 1930s that chicks reared on a reconstituted ‘sterol-free’ diet developed a
hemorrhagic disease characterized by a severe impairment in blood coagulation
(Almquist and Stokstad, 1935; Dam, 1929, 1935; Dam and Schønheyder, 1934;
Holst and Halbrook, 1933; McFarlane et al., 1931; Schønheyder, 1935). The
lack of sterols or fat in the diet asthe cause of the disease was quickly ruled out,
as haemorrhages still appeared in chicks receiving a daily supplement of choles-
terol and oil from cod-liver or flax seeds. Nor did it appear that the haemor-
rhages were caused by a lack of any of the known vitamins.Feeding experiments
with supplements obtained from various fractionation procedures showed,
however, that the protecting factor resembled vitamin E, being thermostable,
fat-soluble and non-saponifiable. (Almquist and Stokstad, 1935; Dam, 1935;
Dam and Schønheyder, 1934). The disease could be prevented or cured by
supplementing the chicks’ diet with various plant or animal products such as
fresh cabbage, dried alfalfa, tomatoes, hemp seeds, putrefied fish meal—but not
fresh—or hog liver fat (Almquist and Stokstad, 1935; Dam, 1935; Dam and
Schønheyder, 1934). Almquist and Stokstad (1935)at the College of Agriculture
of the University of California-Berkeley established early on that the green parts
230 CHLOE
¨VAN OOSTENDE ET AL.
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of the plant ingredients were the sources of the antihemorrhagic factor, and that
it was distinct from chlorophyll and xanthophyll. They also understood that, in
the case of putrefied fish meal, the antihemorrhagic factor originated from the
development of microorganisms (Almquist and Stokstad, 1935). Henrik Dam
at the Biochemical Institute of the University of Copenhagen recognized this
antihemorrhagic factor as a vitamin; he named it ‘vitamin K’, for K was the first
letter in the alphabet that had not been used to designate other vitamins, and
coincidently happened to correspond to the first letter in the word ‘koagulation’
as spelled in Scandinavian (Dam, 1935). A few years later, Edward A. Doisy’s
group at the Laboratory of Biological Chemistry from St. Louis University
School of Medicine purified vitamin K
1
(phylloquinone) from alfalfa, deter-
mined its structure and achieved its chemical synthesis (Binkley et al.,1939;
MacCorquodale et al.,1939a,b;McKeeet al.,1939). Shortly after, vitamin K
2
(menaquinone) was isolated from putrefied fish meal and characterized (Doisy
et al.,1941). The 1943 Nobel Prize in Physiology or Medicine was co-awarded to
Henrik Dam ‘for his discovery of vitamin K’ and to Edward A. Doisy ‘for his
discovery of the chemical nature of vitamin K’. The award did not acknowledge
the pioneering work of Almquist and Stokstad, who co-discovered vitamin K
independently from Dam, or that of Schønheyder, who demonstrated that
vitamin K deficiency impaired blood coagulation. As for plants, one had to
wait until the mid-1980s to find out that phylloquinone participates in the
photosynthetic electron transfer chain and until the past couple of years to
discover that it doubles as an electron acceptor linked to the formation of
disulfide bridges in proteins.
Some readers may also be surprised to learn that until the middle of this
decade—and despite the cardinal role played by phylloquinone in photosyn-
thesis and human nutrition—not much was known about the biosynthesis of
this vitamin. As we will see later, plant biochemists were among the leaders in
the early studies of vitamin K biosynthesis. Unfortunately, as emerged a
general assumption that the biosynthesis of phylloquinone in photosynthetic
organisms was identical to that of menaquinone in facultative anaerobic
bacteria, research on the metabolism of vitamin K in plants virtually ceased
for decades. If it is indeed correct to view the individual steps of phylloqui-
none and menaquinone biosynthesis as similar, the most recent investiga-
tions showed that plants evolved an unprecedented architecture to synthesize
phylloquinone, including extraordinary events of gene fusion and horizontal
gene transfer, split of the pathway between plastids and peroxisomes and
multiple metabolic branch points that link the biosynthesis of phylloquinone
to that of salicylate, tocopherols and chlorophylls. The study of phylloqui-
none biosynthesis in cyanobacteria even led a couple of years ago to the
discovery of a ‘missing’ enzyme of the vitamin K biosynthetic pathway.
VITAMIN K
1
(PHYLLOQUINONE) 231
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This review aims to summarize the current knowledge concerning
the function of vitamin K in vertebrates and oxygenic photosynthetic organ-
isms and its metabolism—emphasizing the most recent advances in under-
standing the phylloquinone biosynthetic pathway and its evolution
in plants—and to point out areas that are still obscure. We refer the reader
to the reviews of Sakuragi and Bryant (2006), Fromme and Grotjohann
(2006) and van der Est (2006) for a detailed coverage of the biosynthesis
and function of phylloquinone in cyanobacteria. We will nonetheless discuss
on occasion recent works concerning the biosynthesis of isoprenoid naphtho-
quinones in cyanobacteria and facultative anaerobic bacteria, as some of the
findings in these organisms could represent paradigms for plants.
II. STRUCTURE AND CHEMISTRY OF VITAMIN K
The term vitamin K encompasses a class of fat-soluble compounds formed
from a naphthoquinone ring attached to a poly-isoprenyl side chain of
variable length and saturation (Fig. 1A). Its main natural forms are vitamin
K
1
(phylloquinone; 2-methyl-3-phytyl-1,4-naphthoquinone) that is found in
plants (Oostende et al., 2008), green algae (Lefebvre-Legendre et al., 2007)
and certain cyanobacteria (Collins and Jones, 1981) and vitamin K
2
(mena-
quinones; 2-methyl-3-(all-trans-polyprenyl)-1,4-naphthoquinone) that is
found in most groups of archaea and bacteria (Collins and Jones, 1981),
the cyanobacterium Gloeobacter violaceus (Mimuro et al., 2005), red algae
(Yoshida et al., 2003) and diatoms (Ikeda et al., 2008). Vitamin K-synthesiz-
ing organisms appear to contain either phylloquinone or menaquinones but
not both. Phylloquinone has a partially unsaturated side chain formed of one
isopentenyl followed by three isopentyl units, while menaquinones have a
fully unsaturated side chain composed of 2–13 isopentenyl units (Fig. 1A).
Menaquinones are often designated as menaquinone-n(MK-n), where n
refers to the number of isopentenyl units in the side chain.
The naphthoquinone ring of vitamin K can exist at different levels of
oxidation, varying from epoxide (the most oxidized) to quinol (the most
reduced) through the intermediate quinone and semi-quinone (Fig. 1B).
The epoxide form is the product of an enzymatic reaction that so far
has been identified only in animal cells. Most of plant and cyanobacterial
phylloquinone is in the quinone form (Oostende et al., 2008; Widhalm et al.,
2009).
While in facultative anaerobic bacteria, menaquinones are often found
lacking the methyl group at position 2 of the naphthoquinone ring—for
instance, up to 90% of the menaquinone pool in aerobically grown
232 CHLOE
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Escherichia coli is demethylated (Unden, 1988)—in plants and cyanobac-
teria, phylloquinone is virtually all in the methylated form [there is a single
report of the presence of trace levels of demethylphylloquinone in spinach
chloroplasts (McKenna et al., 1964)].
III. BIOCHEMICAL ROLES OF VITAMIN K
A. VERTEBRATES
The major known function of vitamin K in vertebrates is that of a cofactor
for the -carboxylation of specific glutamate residues, thus conferring strong
chelating properties to certain proteins whose activity depends on calcium
binding. Among such -carboxylglutamate (Gla)-containing proteins are
Fig. 1. (A) Structures of phylloquinone (vitamin K
1
) and menaquinones
(vitamin K
2
). The phytyl side chain of phylloquinone contains one isopentenyl unit
and three isopentyl units, while that of menaquinones are made exclusively of iso-
pentyl units. (B) Interconversion of the different redox forms of the naphthoquinone
ring. R: poly-isoprenyl moiety.
VITAMIN K
1
(PHYLLOQUINONE) 233
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blood coagulation factors (prothrombin, factors VII, IX and X), proteins
that participate in bone metabolism (osteocalcin, Matrix Gla Protein) and
cell signalling (Gas6).
In order to fulfil its role as cofactor for the -carboxylase, vitamin K must
be in the quinol form. As a by-product of the -carboxylation, the bireduced
naphthalenoid ring of vitamin K is converted to the fully oxidized epoxide
form (Fig. 2). It is salvaged by an integral enzyme complex, named Vitamin
K epoxide reductase (VKOR), whose catalytic subunit (VKORC1; EC
1.1.4.1) reduces the epoxide back to quinone and the quinone back to quinol
(Chu et al., 2006;Fig. 2). This enzyme is the target of the vitamin K antago-
nist warfarin, used as an anticoagulant drug and rodenticide. Besides its role
as a cofactor, studies on mammalian cell cultures indicate that vitamin K acts
as a transcriptional regulator (Ichikawa et al., 2006) and as an antioxidant
(Li et al., 2003).
The lack of vitamin K results in non-functional Gla-containing proteins,
which, in turn, can lead to impaired blood clotting and bone mineralization.
Severe vitamin K deficiency, which can lead to easy bruising and bleeding, is,
however, rare in healthy adults because vitamin K is widespread in foods,
Fig. 2. Scheme of the vitamin K-dependent -carboxylation of glutamyl residues
in vertebrates. Vitamin K quinol is converted to an oxygenated intermediate that
abstracts a proton from the -carbon of the glutamyl residue of the carboxylase
substrate, followed by the addition of carbon dioxide. The concomitant oxidation
of vitamin K quinol into vitamin K epoxide, and its subsequent salvaging by the
enzyme complex vitamin K epoxide reductase (VKOR), is called the vitamin K cycle.
234 CHLOE
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and the gut flora produces basal levels of menaquinones (Suttie, 1995). Only
individuals having chronicle hepatic and pancreatic disorders (Savage and
Lindenbaum, 1983), those receiving long-term antibiotic (Savage and
Lindenbaum, 1983; Shevchuk and Conly, 1990) or vitamin K antagonist
treatments (Bach et al., 1996), appear to be at risk of acute vitamin K
deficiency. Newborns, whose intestinal flora is not yet established, stand
apart and are naturally exposed to an increased risk of vitamin K deficien-
cy—often leading to dramatic haemorrhage of the central nervous system.
The risk is actually higher for infants who are exclusively breast-fed because
the human milk contains only traces of this vitamin (American Academy of
Pediatrics, 2003). It is therefore routine—and often mandatory—in many
countries to administer intramuscular or oral vitamin K at birth as a pro-
phylactic measure (American Academy of Pediatrics, 2003). The incidence of
unexpected bleeding during the first week of life in previously healthy neo-
nates ranges from 250 to 1700 per 100,000 births, and these numbers rise
to 4400–7200 per 100,000 births in infants 2–12 weeks of age who have
received no or inadequate vitamin K prophylaxis (American Academy of
Pediatrics, 2003).
The adequate intake values for vitamin K in the United States are current-
ly set at 120 g/day for men and 90 g/day for women (Food and Nutrition
Board, 2001). Specific levels have not yet been established in the European
Union, but the Committee on Medical Aspects of Food and Nutrition Policy
in the United Kingdom considered that an intake of 1 g/kg of body weight/
day is likely adequate for the proper carboxylation of blood coagulation
factors. In a typical western diet, phylloquinone is the main contributor of
vitamin K intake; about half of it comes from green leafy vegetables, fol-
lowed by soybean, olive, canola and cottonseed oils (Booth and Suttie, 1998).
American and British studies reported average values for dietary vitamin K
intake ranging from 60 to 70 g/day and suggested that one-half of the
populations investigated had vitamin K intakes below the present guidelines
(Vermeer et al., 2004). The impact on bone health of such suboptimal intakes
is currently debated. There is evidence that the level of circulating under-
carboxylated osteocalcin increases after menopause, and that it correlates
with an increased risk of hip fracture (Szulc et al., 1996). Some epidemiologi-
cal studies also reported that individuals with the highest vitamin K intakes
have lower risk of hip fracture than those with the lowest intakes (Booth
et al., 2000; Feskanich et al., 1999), but others found no correlations
(McLean et al., 2006; Rejnmark et al., 2006). None of these studies could
establish a relationship between vitamin K intake and bone mineral density.
Clinical trials indicated a possible increase in bone mineral density and bone
strength in postmenopausal women receiving vitamin K supplementation,
VITAMIN K
1
(PHYLLOQUINONE) 235
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but the doses used were several orders of magnitude higher than those
commonly found in the diet (Iwamoto et al., 2001; Knapen et al., 2007).
B. PLANTS AND CYANOBACTERIA
Until recently, the sole firmly established function of phylloquinone in pho-
tosynthetic organisms was that of a light-dependent electron carrier—the A
1
acceptor—in photosystem I (Brettel et al., 1987; Petersen et al., 1987;
Sigfridsson et al., 1995). The process is a one-electron transfer, that is, a
quinone/semi-quinone turnover—from chlorophyll ato the iron–sulphur
cluster of ferredoxin reductase (Boudreaux et al., 2001; Sigfridsson et al.,
1995;Fig. 3). There are two molecules of phylloquinone, called Q
K
A and
Q
K
B, that are bound to the PsaA and PsaB subunits, respectively, at the
stromal side of each photosystem I monomer (Ben-Shem et al., 2003;
Boudreaux et al., 2001; Jordan et al., 2001). Both molecules are active in
electron transport, but the transfer rate through Q
K
B appears to be 50 times
higher than that through Q
K
A(Guergova-Kuras et al., 2001). The reasons
for such a difference in kinetics between the two branches are not yet fully
understood (Fromme and Grotjohann, 2006).
Fig. 3. Scheme of the electron transfer in photosystem I and approximate mid-
point potentials of the cognate electron carriers in plants and cyanobacteria. Phyllo-
quinone is located at the A
1
site of the PsaA and PsaB subunits of photosystem I to
serve as a one-electron carrier from chlorophyll aA
0
) to the Fe-S cluster (F
X
,F
A
/F
B
).
P700, photosystem I reaction center; P700*, excited photosystem I reaction center.
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Reports that at least half of phylloquinone is not bound to photosystem I
(Gross et al., 2006; Lohmann et al., 2006), together with the detection of the
quinol form of phylloquinone in multiple plant species (Oostende et al., 2008)
and the cyanobacterium Synechocystis (Widhalm et al., 2009), have recently
indicated that in photosynthetic organisms, phylloquinone is probably
involved in redox reactions distinct from that of the one-electron transfer
in photosystem I. Further evidence for such an additional role came with the
discovery of phylogenetic relationships between mammalian VKOR and
predicted oxidoreductases in photosynthetic organisms. Indeed, although
there is no genomic or biochemical indication that the -carboxylation of
glutamic acid residues and the resulting generation of vitamin K epoxide
occur outside of the metazoan lineage, homology searches detect cyanobac-
terial and plant proteins that are similar to mammalian VKORC1. Remark-
ably, these VKORC1 homologues display a C-terminal fusion with a soluble
thioredoxin-like domain having the hallmarks of a protein disulfide isomer-
ase (Furt et al., 2010; Goodstadt and Ponting, 2004; Li et al., 2010; Singh
et al., 2008). In vitro assays demonstrated that the Synechococcus-fused
enzyme could couple the formation of disulfide bonds in an artificial protein
substrate to the reduction of phylloquinone (Li et al., 2010). Similarly, the
Arabidopsis orthologue—the At4g35760 gene product, which is localized in
plastids—was shown to catalyze the conversion of conjugated naphthoqui-
none species into their quinol forms using either dithiotreitol or its protein
disulfide isomerase moiety as electron donors. However, unlike mammalian
VKORC1, the plant and cyanobacterial enzymes lack phylloquinone epoxide
reductase activity and are resistant to warfarin (Furt et al., 2010). The
Arabidopsis enzyme also appears to be inactive on conjugated benzoqui-
nones such as plastoquinone and ubiquinone. Such a substrate stringency
might be unique to plants, for there is evidence that the recombinant Syne-
chococcus enzyme binds ubiquinone (Li et al., 2010), so as does the E. coli
DsbB protein (quinone oxidoreductase), which features similarities in se-
quence, structure and mode of action with VKORC1, and in fact uses
menaquinone or ubiquinone as oxidant molecules (Inaba et al., 2004;
Takahashi et al., 2004).
Along this line, it is noteworthy that in Synechocystis sp. PCC 6803, the
phenotype of the VKORC1-like (slr0565) knockout does not parallel that of
mutant strains lacking phylloquinone. Indeed, deletion of slr0565 causes
lethality or severe growth retardation depending on the presence or absence
of glucose in the culture medium, respectively (Singh et al., 2008), whereas in
similar conditions, Synechocystis mutants blocked in phylloquinone biosyn-
thesisdisplay either no or moderate growth defects (see Section V.G). It is
therefore conceivable that in phylloquinone-deficient cyanobacteria, the
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1
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VKORC1-like enzyme functions using an alternate substrate, possibly plas-
toquinone. In plants, however, the lack of phylloquinone causes complete
loss of photoautotrophy (see Section V.G), and failure to obtain Arabidopsis
transgenics, whose VKORC1-like expression is deregulated, indicating that
this enzyme fulfils a core and vital role (Furt et al., 2010).
IV. DETECTION AND DISTRIBUTION OF
PHYLLOQUINONE IN PLANTS
A. DETECTION
Early methods for the determination of vitamin K in food or biological
extracts relied on a chick bioassay. Analyses were tedious, entailing large
extraction volumes—especially in samples of animal origin due to their
extremely low vitamin K content—and provided only semiquantitative
data (Dam and Schønheyder, 1936). Subsequent quantitative methods were
developed using thin-layer chromatography, gas chromatography and high-
performance liquid chromatography (HPLC); the latter coupled either to
fluorometric or to electrochemical detection (Davidson and Sadowski, 1997;
McCarthy et al., 1997). HPLC methods based on the reduction of the
naphthoquinone ring to its fluorescent quinol form prior to its detection by
fluorometry have proven to combine high sensitivity and selectivity and are
today the preferred applications for the routine quantification of vitamin K
in complex extracts (Booth and Sadowski, 1997; Davidson and Sadowski,
1997). On an additional technical note, let us mention that in green plant
tissues, phylloquinone is often sufficiently abundant to be detected and
quantified using HPLC–spectrophotometry (Fraser et al., 2000).
B. TISSULAR DISTRIBUTION
The level of phylloquinone varies greatly between different plant species and
tissues (Table I). Leaves usually have the highest levels, while most fruits,
tubers and seeds contain several-fold less. It is noteworthy that staple crops
(e.g. grains and tubercles) are among the poorest plant sources of
phylloquinone.
C. SUBCELLULAR DISTRIBUTION
At the subcellular level, plastids account for most if not all of the phylloquinone
content of plant tissues (Lohmann et al.,2006;Oostendeet al.,2008). Subplas-
tidial fractionation experiments demonstrated that about a third of
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phylloquinone in Arabidopsis chloroplasts is deposited in plastoglobules, and
therefore, a significant amount of phylloquinone is not bound to photosystem I
(Lohmann et al.,2006). A similar conclusion was inferred from the observation
that Arabidopsis mutants containing less than a quarter of wild-type levels of
phylloquinone retained most of their photosystem I activity (Gross et al.,2006).
The enrichment of naphthoquinone oxidoreductase activities in plasma
membrane preparations of corn roots (Lu
¨thje et al., 1998) and soybean
hypocotyls (Bridge et al., 2000; Schopfer et al., 2008) suggests that small
pools of phylloquinone may occur outside plastids. One of these studies
reported the direct detection of phylloquinone in the plasma membrane
(Lu
¨thje et al., 1998), but the possibility of proplastid breakage (e.g. using
galactolipids as markers) was not investigated.
TABLE I
Phylloquinone Content of Some Plant Species and Plant Food-Products
Phylloquinone (g/100 g)
A. thaliana (green leaf) 365
(a)
Brassica oleracea (canola oil) 127
(b)
Brassica oleracea (collard greens) 440
(b)
Brassica oleracea (broccoli) 180
(b)
Brassica oleracea (brussel sprouts) 177
(b)
Brassica oleracea (cauliflower) 20
(b)
Cicer arietinum (chickpeas) 9
(c)
Daucus carota (tuber) 2.7
(a)
Lactuca sativa (green leaf) 126
(c)
Lactuca sativa (‘iceberg’ lettuce) 35
(b)
Manihot esculenta (cassava) 1.9
(c)
Olea europaea (olive oil) 55
Oryza sativa (grain) 0.1
(c)
Oryza sativa (green leaf) 662
(a)
Phaseolus vulgaris (dry bean) 5.6
(c)
Phaseolus vulgaris (green beans) 33
(b)
Solanum. lycopersicon (green leaf) 1217
(a)
Solanum. lycopersicon (green fruit) 19
(a)
Solanum. lycopersicon (red ripe fruit) 8
(a)
Solanum tuberosum (tuber) 1.3
(a)
Glycine max (soybean oil) 193
(b)
Glycine max (‘Edamame’ seed) 31
(c)
Triticum spp. (whole grain flour) 1.9
(c)
Vicia faba (fava bean) 9
(c)
Zea mays (grain) 0.3
(c)
Zea mays (green leaf) 1514
(a)
Zea mays (oil) 3
(b)
Data are compiled from Oostende et al. (2008)
(a)
;Booth and Suttie (1998)
(b)
; USDA National
Nutrient Database for Standard Reference (http://www.nal.usda.gov/fnic/foodcomp/search/)
(c)
.
Staple crops are shown in bold.
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V. PHYLLOQUINONE BIOSYNTHESIS IN PLANTS
In essence, the phylloquinone biosynthetic pathway of plants consists of two
separated metabolic branches: one for the naphthoquinone ring and the
other for the phytyl moiety, which is also used for the biosyntheses of
tocopherols and chlorophylls. We focus hereafter on the enzymatic steps
leading to the formation of the naphthoquinone ring, as the biosynthesis of
the phytyl-diphosphate precursor from the methylerythritol-phosphate path-
way in plastids is not specific to phylloquinone and has been previously
covered (see, for instance, Lichtenthaler, 1999; Rohmer, 2003).
The biosynthesis of the naphthoquinone ring entails seven enzymatic steps.
The immediate precursor chorismate is first converted into isochorismate, to
which a succinyl side chain is added at the C2 position (Fig. 4). After
elimination of pyruvate and aromatization of the cyclohexadiene ring, the
succinyl chain is activated by ligation with CoA and then cyclized, yielding
1,4-dihydroxynaphthoyl-CoA (DHNA-CoA). The CoA moiety is then
removed, DHNA is conjugated to its phytyl partner and then methylated.
An alternative pathway, in which the naphthoquinone backbone originates
from a chorismate–inosine conjugate termed futalosine, was recently
described in some species of Deinococcus-Thermus, Actinobacteria and
E-Proteobacteria (Hiratsuka et al., 2008). There is currently no genomic or
biochemical evidence that this biosynthetic route occurs in phylloquinone-
synthesizing eukaryotes.
A. EARLY WORK
The basic architecture of isoprenyl naphthoquinone biosynthesis was estab-
lished in the 1970s and 1980s simultaneously in plants and facultative anaer-
obic bacteria using radiolabelling experiments. It quickly emerged from these
studies that the individual steps of the phylloquinone and menaquinone
biosynthetic pathways were virtually identical. In plants, shikimate was
identified as the precursor of the naphthoquinone moiety via the formation
of o-succinylbenzoate (OSB), OSB-CoA and DHNA (Dansette and Azerad,
1970; Heide et al., 1982; Hutson and Threlfall, 1980; Thomas and Threlfall,
1974), while the ring prenylation and methylation steps were found to use
phytyl-diphosphate and s-adenosylmethionine as substrates, respectively
(Gaudillie
`re et al., 1984; Schultz et al., 1981). Interestingly, some of these
early works revealed that in Rubiaceae, OSB doubles as an intermediate in
the biosynthesis of anthraquinone species—of which still today not much is
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known—and suggested that anthraquinone and naphthoquinone biosynth-
eses intersect at the level of DHNA.
The plant genes of phylloquinone biosynthesis have been identified only in
the past decade; for most of them using the genomic and genetic resources of
Arabidopsis thaliana and homology searches with the bacterial men genes as
query. Table II lists the cognate Arabidopsis proteins with their orthologues
in Synechocystis sp. PCC 6803 and E. coli. Two steps (reactions 6 and 7,
Fig. 2) remain to be characterized: for the first one, the gene is predicted but
has not been functionally confirmed; for the second one, orthology appears
to be missing (see below).
Fig. 4. The biosynthesis pathway of phylloquinone. 1, isochorismate synthase;
2, SEPHCHC synthase; 3, SHCHC synthase; 4, OSB synthase; 5, OSB-CoA ligase;
6, DHNA-CoA synthase; 7, DHNA-CoA thioesterase; 8, DHNA prenyltransferase;
9, demethylphylloquinone methyltransferase. SAH, s-adenosylhomocysteine;
SAM, s-adenosylmethionine; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-
cyclohexene-1-carboxylic acid; SHCHC, (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohex-
adiene-1-carboxylic acid. EC numbers are indicated under each corresponding
reaction.
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B. ISOCHORISMATE SYNTHASE/PHYLLO (REACTIONS 1–4)
Genetic approaches identified an Arabidopsis gene, termed PHYLLO
(At1g68890), that encodes an extraordinary protein composed of four mod-
ules homologous to the bacterial MenF (5.4.4.2), MenD (2.2.1.9), MenC
(4.2.1.113) and MenH (4.2.99.20) proteins, respectively (Fig. 5). The MenF
module lacks a complete chorismate binding domain, suggesting that it
cannot catalyze the conversion of chorismate into isochorismate (this
truncated domain appears nevertheless to be conserved from monocots to
dicots, pointing to a selective driving force for its maintenance. What this is,
however, is still an enigma). The Arabidopsis genome encodes in fact two
separated and catalytically active isochorismate synthases, ICS1 (At1g74710)
and ICS2 (At1g18870), that share about 80% identity (Garcion et al., 2008;
Strawn et al., 2007; Wildermuth et al., 2001). The ics1/ics2 double knockout
is devoid of phylloquinone (Garcion et al., 2008; Gross et al., 2006), thus
providing genetic evidence that PHYLLO is not sufficient for the de novo
synthesis of isochorismate, and that phylloquinone biosynthesis is dependent
upon a pool of isochorismate that is produced by separated isochorismate
synthases. As ICS1 bears most of the flux of isochorismate biosynthesis
(Garcion et al., 2008; Gross et al., 2006) and several plant genomes encode
for a single ICS copy, it is unclear if Arabidopsis ICS1 and ICS2 have
dedicated functions, or simply originate from recent duplication events and
are evolving separately. Whatever the case, isochorismate represents a meta-
bolic branch point where plant phylloquinone biosynthesis is likely to
TABLE II
Correspondence Between the Phylloquinone Biosynthesis Enzymes in Arabidopsis
and Synechocystis and Their Orthologues Involved in the Biosynthesis of
Menaquinone-8 in E. coli
A. thaliana
Synechocystis sp.
PCC6803 E. coli
Isochorismate synthase At1g74710 (ICS1)
At1g18870 (ICS2)
Slr0817 MenF
SEPHCHC synthase At1g68890 (PHYLLO) Sll0603 MenD
SHCHC synthase At1g68890 (PHYLLO) Slr1916 MenH
OSB synthase At1g68890 (PHYLLO) Sll0409 MenC
OSB-CoA ligase At1g30520 (AAE14) Slr0492 MenE
DHNA-CoA synthase At1g60550 (putative) Sll1127 MenB
DHNA-CoA thioesterase Unknown Slr0204 Unknown
DHNA phytyltransferase At1g60600 (ABC4) Slr1518 MenA
Demethylphylloquinone
methyltransferase
At1g23360 Sll1653 UbiE
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compete with that of the plastid-produced hormone, salicylate, which also
uses isochorismate as a precursor (Wildermuth et al., 2001). Confirming this
view, the constitutive expression in tobacco chloroplasts of a bacterial iso-
chorismate pyruvate lyase, which converts isochorismate to salicylate, was
shown to result in an increase in salicylate level at the expense of phylloqui-
none (Verberne et al., 2007). At the time of the identification of PHYLLO,
MenH was still thought to correspond to DHNA-CoA thioesterase. The
menC and menH fused modules were therefore viewed as encoding for
domains that catalyzed reactions two steps apart from each other. It was
hypothesized that such an arrangement could indicate the existence of physi-
cal associations between PHYLLO, OSB-CoA ligase and DHNA synthase
(Gross et al., 2006). It is now evident that the PHYLLO MenDCH modules
catalyze consecutive reactions that lead to the synthesis of OSB. This does
not actually rule out that PHYLLO interacts with other enzymes in the
pathway, particularly because the fused structure of PHYLLO itself is sug-
gestive of a metabolon where biosynthetic intermediates are channelled from
one catalytic domain to the other.
Homology searches point to the existence of clusters of menF, menD,
menC and menH orthologues in green algae, mosses, diatoms and rhodo-
phytes. The menF domain of such clusters, in contrast to that of flowering
plants, features a full chorismate binding domain and is a priori functional.
AChlamydomonas reinhardtii cDNA corresponding to the menD orthologue
was shown to contain in-frame stop codons both upstream of the initiation
codon and at the end of the coding sequence, suggesting that in this species,
the menF, menD and menC modules are translated as separated polypeptides
(Lefebvre-Legendre et al., 2007). It remains therefore to establish if fused and
multifunctional enzymes equivalent to PHYLLO occur outside of the flower-
ing plant lineage.
Fig. 5. Arrangement of the functional domains of the Arabidopsis PHYLLO
protein and their approximate percentage of identity with their separated Men
orthologues in E. coli and with Arabidopsis isochorismate synthases 1 and
2 (AtICS1 and AtICS2). CTP, chloroplast transit peptide.
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C. OSB-COA LIGASE (REACTION 5)
OSB-CoA ligase (6.2.1.26) activates the carboxyl group on the succinyl side
chain of OSB by creating a high-energy bond with the pantetheine moiety of
CoA (Kolkmann and Leistner, 1987;Fig. 2). Plant OSB-CoA ligase was
identified as part of a general characterization effort of the CoA ligase family
in Arabidopsis (Kim et al., 2008). A putative CoA ligase termed AAE14 (acyl
activating enzyme 14; the product of gene At1g30520) was singled out as one
of the top coexpressors of some previously identified phylloquinone biosyn-
thetic genes (At1g60600, DHNA phytyl transferase; At1g23360, demethyl-
phylloquinone methyltransferase; At1g68890, PHYLLO) and of the
predicted DHNA-CoA synthase (At1g60550; see below). Direct evidence
for the involvement of AAE14 in phylloquinone biosynthesis came from
the isolation of three independent T-DNA mutant lines corresponding to
insertions in the first intron, and fourth and ninth exons of At1g30520,
respectively; all of which lacked phylloquinone (Kim et al., 2008). The
T-DNA mutants were also found to accumulate OSB and could be partially
rescued by exogenous applications of DHNA (Kim et al., 2008). Expression
of At1g30520 cDNA was shown to fully restore menaquinone biosynthesis in
the E. coli menE knockout, thus verifying that AAE14 bore OSB-CoA ligase
activity (Kim et al., 2008).
D. DHNA-COA SYNTHASE/DHNA-COA THIOESTERASE (REACTIONS 6/7)
DHNA-CoA synthase (4.1.3.36) catalyzes the cyclization of OSB-CoA
(Fig. 2). The enzyme, which belongs to the crotonase-fold family, is often
termed in the literature and numerous databases as DHNA synthase or
naphthoate synthase, but it is clear that its reaction product is DHNA-
CoA, not DHNA (Jiang et al., 2010; Truglio et al., 2003). The enzyme’s
substrate, OSB-CoA, is highly unstable at physiological pH and has been
shown to spontaneously decompose in vitro into the spirodilactone form of
OSB (Fig. 6)(Heide et al., 1982; Meganathan and Bentley, 1979). Should
such a decomposition occur in vivo, it is not known if or how OSB spirodi-
lactone is recycled.
Bacterial DHNA-CoA synthases appear to fall into two catalytic classes.
Type I enzymes use a bound bicarbonate anion as a catalytic base, while type
II enzymes use the side-chain carboxylate of one of their acidic residues
(Jiang et al., 2010). Type I enzymes are consequently deemed bicarbonate
dependent and their type II counterparts bicarbonate independent (Jiang
et al., 2010). Sequence comparisons and phylogenetic reconstructions indi-
cate that cyanobacterial DHNA-CoA synthase and its predicted Arabidopsis
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homologue belong to the type I category, suggesting that phylloquinone
biosynthesis is regulated by the intracellular level of bicarbonate.
The subsequent removal of CoA from DHNA, catalyzed by DHNA-CoA
thioesterase (3.1.2.-), has long puzzled the elucidation of the vitamin K
biosynthesis pathway. After being misattributed to DHNA-CoA synthase
(MenB), then to SHCHC synthase (MenH) in E. coli, it was later proposed
that the hydrolysis of DHNA-CoA, which like its OSB-CoA precursor
spontaneously decomposes at physiological pH, could be merely chemical
(Sakuragi and Bryant, 2006). But recent phylogenomics approaches in cya-
nobacteria detected putative CoA thioesterases, whose encoding genes were
arranged in clusters with known phylloquinone biosynthetic genes (Widhalm
et al., 2009). Deletion of the Synechocystis orthologue—gene slr0204—
resulted in a dramatic decrease of the phylloquinone content in the knockout
cells, thus verifying the existence of a functional linkage between the putative
CoA thioesterase and phylloquinone biosynthesis (Widhalm et al., 2009).
Further investigations demonstrated that the knockout mutant accumulated
DHNA-CoA and could be chemically rescued with DHNA, but not with
OSB, thus pointing to the location of the blockage in the pathway (Widhalm
et al., 2009). The purified recombinant Slr0204 was shown to catalyze the
hydrolysis of DHNA-CoA and to display absolute preference for this sub-
strate. It is thought that such a substrate stringency may reflect the presence
of OSB-CoA upstream in the pathway, and whose enzymatic hydrolysis
would create a futile cycle in the phylloquinone biosynthesis pathway
(Widhalm et al., 2009). Although the Synechocystis slr0204 knockout
completely lacked DHNA-CoA thioesterease activity, low levels of phyllo-
quinone could still be detected in this mutant revealing the occurrence of a
basal chemical hydrolysis of DHNA-CoA in vivo (Widhalm et al., 2009).
Such a background decomposition likely explains why DHNA-CoA
Fig. 6. Hydrolysis and lactonization of OSB-CoA.
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thioesterase did not show up in forward genetic screens aimed at identifying
men genes in bacteria, and one can therefore grasp through this illustrative
case the power of phylogenomics in predicting gene function based on the
detection of conserved physical associations of genes in genomes.
Except for the extremophilic rhodophytes Cyanidiales (Cyanidioschyzon
merolae and Cyanidium caldarium) and the cercozoan (Paulinella chromato-
phora), the plastid or chromatophore of which encode homologues of cya-
nobacterial DHNA-CoA thioesterase arranged in clusters with
phylloquinone biosynthetic genes (see Section VI), DHNA-CoA thioesterase
remains elusive in phylloquinone-synthesizing eukaryotes. Homology
searches do detect two pairs of Arabidopsis paralogs (At1g68260/
At1g68280, At1g35250/At1g35290) that share 17–28% of identity with Syne-
chocystis Slr0204, but these Arabidopsis genes have recently been shown to
encode orthologues of solanaceous methyl ketone synthases (Yu et al., 2010).
E. DHNA PHYTYL TRANSFERASE (REACTION 8)
DHNA phytyl transferase (2.5.1.-), an integral membrane protein, couples
the naphthoquinone ring to the phytyl side chain (Fig. 2). It was the first
enzyme specific to phylloquinone biosynthesis to be described in plants and
was initially identified in the Arabidopsis abc (aberrant chloroplast develop-
ment) T-DNA mutant series (Shimada et al., 2005). The cognate mutant-
designated abc4was shown to correspond to an insertion in the ninth exon of
gene At1g60600 and to lack phylloquinone (Shimada et al., 2005). Function-
al assignment was based on homology with Synechocystis sp. PCC 6803
DHNA phytyl transferase, which shares 41% identity with the At1g60600
protein (Shimada et al., 2005).
F. DEMETHYLPHYLLOQUINONE METHYLTRANSFERASE (REACTION 9)
Demethylphylloquinone methyltransferase (2.1.1.-) catalyzes the methyla-
tion of 2-phytyl-1,4-naphthoquinone (demethylphylloquinone) and corre-
sponds to the last step of the phylloquinone biosynthetic pathway (Fig. 2).
Mining the Arabidopsis genome with Synechocystis demethylphylloquinone
methyltransferase—the E. coli UbiE homologue, which doubles in the bio-
synthesis of ubiquinone, hence its name (Lee et al., 1997)—as query detected
the product of gene At1g23360 as a likely orthologue (Lohmann et al., 2006).
Expression of the cognate cDNA fully rescued phylloquinone biosynthesis in
the Synechocystis demethylphylloquinone methyltransferase knockout
(Lohmann et al., 2006). In parallel, a T-DNA line corresponding to an
insertion in the seventh exon of At1g23360 was found to be devoid of
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phylloquinone and to accumulate 2-phytyl-1,4-naphthoquinone, thus estab-
lishing definite evidence that this gene encodes the only demethylphylloqui-
none methyltransferase in Arabidopsis (Lohmann et al., 2006).
G. MUTANT PHENOTYPE
Arabidopsis lines corresponding to phyllo (the fused SEPHCHC synthase–
SHCHC synthase–OSB synthase), aae14 (OSB-CoA ligase) and abc4
(DHNA phytyl transferase) knockouts and to the double knockout ics1/
ics2 (isochorismate synthase 1 and 2) display loss of photoautotrophy and
are seedling lethal (Gross et al., 2006; Kim et al., 2008; Shimada et al., 2005).
A few pale-green leaves can be obtained from these mutants providing that
they are grown on a medium containing sucrose and under low illumination,
but even so the plants eventually stop developing. Analyses of the phyllo and
abc4 mutants showed that the lack of phylloquinone results in the disruption
of photosystem I assembly (Gross et al., 2006; Shimada et al., 2005). Plasto-
quinone level and photosystem II activity were also shown to be dramatically
reduced in the abc4 knockout, while photosystem II was found to be only
moderately affected in the phyllo mutant (Gross et al., 2006; Shimada et al.,
2005). In contrast, green algal and cyanobacterial mutants, which are
blocked in the formation of the naphthoquinone ring or its prenylation, are
able to recruit plastoquinone into the A
1
site of photosystem I in place of
phylloquinone and—though being sensitive to high light intensity—can grow
photoautotrophically (Johnson et al., 2000; Lefebvre-Legendre et al., 2007).
The demethylphylloquinone methyltransferase knockout is the sole viable
phylloquinone-deficient mutant in plants. The reduction in photosynthetic
efficiency and number of photosystem I subunits observed in the cognate
Arabidopsis insertion line indicate nonetheless that the replacement of
phylloquinone by demethylphylloquinone is not fully functional (Lohmann
et al., 2006).
H. SUBCELLULAR LOCALIZATION OF PHYLLOQUINONE
BIOSYNTHETIC ENZYMES
Early radiolabelling experiments showed that the prenylation and methylation
steps of plant phylloquinone biosynthesis were associated with the chloroplast
membranes; the two activities appeared to be localized in separate subfractions:
the prenylation occurring in the chloroplast envelope and the methylation in
thylakoids (Gaudillie
`re et al., 1984; Kaiping et al., 1984; Schultz et al.,1981).
Cloning of Arabidopsis DHNA phytyl transferase and demethylphylloquinone
methyl transferase later confirmed that both enzymes possess N-terminal
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signalling peptides and are indeed targeted to plastids (Lohmann et al., 2006;
Shimada et al.,2005). Similar findings were obtained for isochorismate synthase
1and2(Garcion et al., 2008; Strawnet al., 2007), PHYLLO (Gross et al.,2006)
and OSB-CoA ligase (Kim et al.,2008). Contrasting with this apparent all-
plastidial localization of the pathway, predicted DHNA-CoA synthases from
dicots and monocots have N-terminal extensions that contain a canonical
peroxisomal targeting signal type 2 (RLx
5
HL) and proteomic approaches
have identified the putative Arabidopsis enzyme and its spinach orthologue in
purified peroxisomes (Babujee et al., 2010; Reumann et al.,2007). Expression in
onion epidermal cells of the Arabidopsis proteinfused at its C-terminal end to a
fluorescent reporter protein further verified that the resulting construct was
imported into peroxisomes (Babujee et al.,2010). The green alga C. reinhardtii
and moss P. patens orthologues, however, lack a peroxisomal targeting signal,
so as do their cyanidiale C. merolae and C. caldarium and cercozoan P. chro-
matophora counterparts, which are chloroplast or chromatophore encoded,
thus indicating that the targeting of DHNA-CoA synthase to peroxisome is
not ubiquitous in phylloquinone-synthesizing eukaryotes.
Interestingly, the preceding enzyme, OSB-CoA ligase, displays in most
monocotyledonous and dicotyledonous species a predicted peroxisomal target-
ing signal. In this case, it corresponds to a C-terminal tripeptide (SSL, SNL,
SRL or SKL depending on the species) that typifies a peroxisomal targeting
signal type 1 (Babujee et al.,2010). N-terminally fused fluorescent versions of
Arabidopsis OSB-CoA ligase (AAE14) or of its last 10 residues containing the
SSL signal were expressed in onion epidermal cells and confirmed here again
that the hybrid proteins were targeted to peroxisomes (Babujee et al.,2010).
These exciting observations imply that the activation of OSB and its cyclization
into DHNA-CoA occur in peroxisomes, thus requiring the shuttling of phyllo-
quinone biosynthetic precursors in and out of plastids and peroxisomes. One
should note, however, that transient expressions of C-terminally tagged fluo-
rescent versions of AAE14 or its first 120 residues in Arabidopsis leaf proto-
plasts and tobacco leaf mesophyll cells, respectively, have demonstrated that the
enzyme also bears a functional plastid targeting presequence and is targeted to
chloroplasts (Kim et al., 2008). The obvious bias of each of the aforementioned
fusion strategies is that the reporter protein conceals either the peroxisomal
targeting signal type 1 (C-terminal fusion) or the plastid targeting presequence
(N-terminal fusion). Although the current view is that AAE14 could be dual
targeted, direct identification using proteomics approaches and/or assays of
OSB-CoA ligase in purified peroxisomes and chloroplasts is needed to precisely
determine the subcellular localization of this enzyme.
As for the hydrolysis of DHNA-CoA, preliminary data from our labora-
tory indicated that although DHNA-CoA thioesterase was detectable in
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whole extracts of pea and Arabidopsis leaves, it was absent in the
corresponding preparations of purified chloroplasts (unpublished results).
As previously suggested (Babujee et al., 2010), this makes the localization of
DHNA-CoA hydrolysis in peroxisomes all the more likely. Figure 7 sum-
marizes the current knowledge concerning the subcellular compartmentation
of the phylloquinone biosynthesis pathway in Arabidopsis.
Fig. 7. Subcellular localization of the phylloquinone biosynthetic enzymes in
Arabidopsis. Letters in brackets specify the type of experimental evidence: fusion to
a fluorescent reporter protein and transient expression in Arabidopsis leaf or leaf
protoplasts (a), onion epidermal cell (b), tobacco mesophyll cells or leaf protoplasts
(c); C-terminal fusion to V5-6xHis epitope and stable expression under the control of
native promoter in Arabidopsis transgenics, and immunolocalization (d); in vitro
import assay in chloroplasts purified from pea seedling (e); subcellular fractionation
and identification using mass sprectrometry (f). Dashed arrows indicate putative
transport steps between plastid and peroxisome, or the possible occurrence of
DHNA-CoA hydrolysis in peroxisome (reaction 7). Evidence from our laboratory
indicates that DHNA-CoA thioesterase activity is lacking in chloroplasts
(Widhalm J.R., unpublished data).
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VI. EVOLUTION OF NAPHTHOQUINONE
BIOSYNTHESIS IN PHOTOSYNTHETIC EUKARYOTES
Isoprenoid naphthoquinones are evolutionarily the most ancient of all con-
jugated quinones, their biosynthesis in prokaryotes predating the rise of
atmospheric dioxygen level ca. 2.5 billion years ago (Schoepp-Cothenet
et al., 2009). Menaquinones have thus been detected in most prokaryotic
lineages (Collins and Jones, 1981), rhodophytes (Yoshida et al., 2003) and
diatoms (Ikeda et al., 2008). Phylloquinone in contrast appears to be restrict-
ed to some cyanobacterial species, green algae and plants (Collins and Jones,
1981; Lefebvre-Legendre et al., 2007; Oostende et al., 2008).
All the phylloquinone biosynthetic enzymes identified so far in photosyn-
thetic eukaryotes are nuclear encoded. However, the cyanidiale orthologues—
with the exception of the MenG orthologue (see below)— are plastid encoded,
indicating that the cognate genes have likely been retained from the former
cyanobacterial endosymbiont. While it might therefore seem that such genes
are merely of direct cyanobacterial descent, some phylogenetic studies suggest
that this is not so for the menF, menD, menC, menE and menB orthologues,
which are in fact more closely related to the chlorobi/-proteobacteria lineage
(Gross et al., 2008). To reconcile this surprising genealogy with the
fact that the corresponding enzymes are plastid encoded in cyanidiales, it
has been proposed that men genes originating from an organism of the
chlorobi/-proteobacteria descent have been captured through horizontal
gene transfer by the free living cyanobacterial progenitor of plastids, that is,
prior to its endosymbiosis, and have replaced their pre-existing cyanobacterial
counterparts (Gross et al., 2008). The remnant of this horizontal gene transfer
would now ‘survive’ as a men gene cluster in the plastid genome of modern-day
cyanidiales and in the nuclear genome of diatoms, green algae and plants—the
aforementioned tetramodular PHYLLO locus (Gross et al., 2008).
In contrast, the cyanidiales menA homologue—also part of such a men
gene cluster—would have been acquired through an independent horizontal
gene transfer with a prokaryotic donor, whose identity remains unclear
(Gross et al.,2008). So is the case for the menA homologue of diatoms,
which would define another event of horizontal gene transfer that occurred in
the nucleus (Gross et al., 2008). As for the nuclear-encoded menG homo-
logue, phylogenies suggest that it would originate from -proteobacteria in
both cyanidiales and diatoms (Gross et al., 2008). Plants and green algae
further complicate the picture, having menE homologues that would branch
from the -proteobacteria lineage and menA and menG homologues that
would be of cyanobacterial ancestry (Gross et al., 2008). In essence, accord-
ing to such phylogenetic reconstructions, the eukaryotic genes involved in the
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formation of isoprenoid naphthoquinones display a high degree of evolu-
tionary chimerism that varies with the lineage considered, owing to multiple
and unrelated events of horizontal gene transfer and/or gene losses (summar-
ized in Fig. 8).
Fig. 8. Tentative scenario for the evolution of the men genes in photosynthetic
eukaryotes as proposed by Gross et al. (2008). Arrows symbolize gene transfers. Note
that menH homologues are not detected in the plastid genomes of cyanidiales. As for
menA, the cyanobacterial progenitor of plastids would have harboured two cognate
homologues: menA1, of cyanobacterial descent and menA2, acquired by HGT from
an unknown prokaryotic donor. Cyanidiales would have lost menA1 and retained
menA2; the opposite would have happened in plants and green algae. An alternative
explanation would be that menA1 was acquired from cyanobacteria by direct HGT in
the nucleus of the green algal ancestor. HGT, horizontal gene transfer.
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One should, however, point that comparative genomics of modern cyano-
bacteria, cyanidiales and chlorobi/-proteobacteria question some parts of this
evolutionary model. For instance, the genomic organization of the men and
DHNA-CoA thioesterase homologues of certain present-day cyanobacteria
and of cyanidiales displays striking similarities that are not conserved in the
chlorobi/-proteobacteria lineage (Fig. 9). Although such a conservation might
be purely coincidental or driven by identical selective constraints (e.g. transcrip-
tional regulation), it could also point to an overlooked phylogenetic closeness
between the menaquinone biosynthetic genes of cyanidiales and some of their
homologues involved in phylloquinone biosynthesis in cyanobacteria.
Fig. 9. Organization of the phylloquinone/menaquinone biosynthetic gene clus-
ters in representative species of cyanobacteria/cercozoan, cyanidiales, -proteobac-
teria and chlorobi. The dashed frame highlights the conserved arrangement of the men
and DHNA-CoA thioesterase (THIO) homologues in cyanobacteria (N. punctiforme,
Nostoc punctiforme;P. marinus,Prochlorococcus marinus; S. sp. CC9605, Synechoc-
coccus sp. CC9605), cercozoan (Cerc.) (P. chromatophora,Paulinella chromatophora)
and cyanidiales (C. caldarium,Cyanidium caldarium;C. merolae,Cyanidioschyzon
merolae). The gene cluster of the cercozoan species P. chromatophora is located in a
plastid-like organelle called the chromatophore; the later is thought to originate from
a recent endosymbiosis of a cyanobacterium of the Prochlorococcus/Synechococcus
lineage. A. hydrophila,Aeromonas hydrophila;C. limicola,Chlorobium limicola;
C. ferrooxidans,Chlorobium ferrooxidans.
252 CHLOE
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VII. PHYLLOQUINONE TURNOVER
Our knowledge of vitamin K metabolism is almost exclusively restricted to
mammals and is largely extrapolated from the data obtained with tocopher-
ols. Pharmacological studies have shown that phylloquinone is rapidly cat-
abolized into shortened side-chain carboxylic acids, which are excreted in
urine as water-soluble glucuronic acid conjugates (Harrington et al., 2007;
Landes et al., 2003). The enzymatic reactions that lead to the shortening of
the side chain are not known sensu stricto. Nevertheless, as tocopherols and
phylloquinone share the same phytyl side chain and in mammals the pro-
ducts of tocopherol catabolism come from !-hydroxylation and subsequent
-oxidation of the side chain, it is believed that phylloquinone follows a
similar catabolic route (Harrington et al., 2007; Landes et al., 2003).
Close to nothing is known about the catabolism of vitamin K in plants. One
study showed that non-physiological doses of phylloquinone can be fed to pea
stem sections, and that more than 90% of the incorporated vitamin could be
recovered after 18-h incubation (Gaunt and Stowe, 1967). The occurrence of
degradation products of phylloquinone in plants is not documented.
VIII. ENGINEERING OF PHYLLOQUINONE
IN PLANTS
There has not been so far any dedicated engineering of phylloquinone in
plants; the only data available are for tobacco transgenics engineered for
salicylic acid biosynthesis, and functional complementation experiments in
Arabidopsis. Thus, in tobacco, the overexpression of an E. coli isochorismate
synthase targeted to plastids led to a fourfold increase of phylloquinone
above wild-type levels (Verberne et al., 2007). In Arabidopsis, overexpression
of demethylphylloquinone methyltransferase or OSB-CoA ligase did not
change phylloquinone content compared to that of wild-type plants (Kim
et al., 2008; Lohmann et al., 2006).
IX. CONCLUDING REMARKS
Findings from the most recent studies of phylloquinone metabolism in plants
illustrate once more that the architecture of plant secondary metabolism is
hardly inferable from previous work in microorganisms. Witness the unprec-
edented and extraordinary multifunctional PHYLLO, the occurrence of
functional redundancies (isochorismate synthases), the apparent lack of
VITAMIN K
1
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orthology (DHNA-CoA thioesterase) and the split of phylloquinone biosyn-
thesis between plastids and peroxisomes.
Besides the identification of the ‘missing’ DHNA-CoA thioesterase and
the characterization of DHNA-CoA synthase—especially with regards to its
possible regulation by carbonate—one of the priorities of the research on
phylloquinone biosynthesis in plants is now to determine the arrangement of
the plastid and peroxisomal branches that lead to the formation of the
naphthoquinone ring. One cannot indeed overemphasize that the cognate
transport steps between these two organelles are as determinant for the flux
of phylloquinone production as the biosynthetic enzymes themselves. It will
therefore need to be established which biosynthetic intermediates are trans-
ported, and if specific transporters are involved. Such future investigations
are predictably challenging owing to the very low abundance and high
instability of most of the naphthoquinone ring’s biosynthetic intermediates,
and to the difficulty inherently attached to the isolation of reasonably pure
plant organelles and to the functional study of integral proteins.
Another area to further explore is the integration of phylloquinone in the
metabolic network of plastids. As mentioned earlier, there is experimental
evidence in Arabidopsis and tobacco that the biosynthetic pathways of
phylloquinone and salicylic acid intersect through isochorismate. It is prob-
able that plants tightly regulate this metabolic node because salicylate is
massively produced in response to certain stresses, while phylloquinone is
absolutely needed as a redox cofactor. One fascinating hypothesis could be
that the flux of isochorismate usage towards phylloquinone biosynthesis
depends on a—yet-to-be demonstrated— physical association between iso-
chorismate synthase and the multifunctional PHYLLO, thus creating a
metabolon from chorismate up to OSB. Such a scenario could also explain
why flowering plants have maintained a truncated and catalytically inactive
MenF domain in PHYLLO; it could serve for instance as a recognition/
binding domain to assemble the metabolon.
Although species specific, the flux split at the level of DHNA between the
naphthoquinone and anthraquinone biosynthetic pathways is even more
enigmatic. Here, again one can expect that anthraquinone-producing species
must have evolved strategies to commit a steady flux of DHNA towards
phylloquinone biosynthesis.
Through phytyl-diphosphate as a common precursor of the isoprenyl
side chain, we also know that phylloquinone is connected to the metabolism
of tocopherols and chlorophyll. However, we cannot currently tell to
what extent a change in the biosynthesis flux of one of this compound will
impact the others. Answering this question is important for the basic under-
standing of the metabolic network of plastid isoprenoids as well as for
254 CHLOE
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engineering purposes. For instance, an increase in phylloquinone level that
would occur at the expense of chlorophyll and/or tocopherols could nega-
tively impact photosynthesis or paradoxically decrease the nutritional value
of the derived plant products. The remark stands for the engineering of
tocopherol levels.
As for the fused VKORC1-PDI enzyme, the research problem is now to
identify the proteins whose folding is connected to the reduction of phyllo-
quinone in plastids. The fate of the formed quinol is also intriguing because
the phylloquinone/phylloquinol ratio appears to be remarkably stable in
plants and Synechocystis (Oostende et al., 2008; Widhalm et al., 2009).
A tacit conclusion is that phylloquinol is re-oxidized, and that these organ-
isms can sense the redox status of their phylloquinone pool.
ACKNOWLEDGEMENTS
G. J. B. and C. v. O. dedicate this chapter to the memory of Dr. Philippe
Raymond, whose mentoring and support have been seminal to their works.
Research in our laboratory is made possible in part by National Science
Foundation Grant MCB-0918258 to G. J. B. and by startup funds provided
by the Center for Plant Science Innovation and the Nebraska Tobacco
Settlement Biomedical Research Development Funds.
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