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Folate Biosynthesis - Hanson
Folate Biosynthesis - Hanson
V I E W
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S
Review in Advance first posted online
on January 26, 2011. (Changes may
C E
I N
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D V A
4.1
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of dihydrofolate 5,10-methylene-THF
the tail promotes enzyme binding and folates
are far more stable to oxidative cleavage when N5–CHO 5-formyl-THF
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Mitochondrion
HMDHP 1 HMDHP [Glycosides]
Plastid F C
HMDHP-P2 DHM DHN
pABA pABA pABA
G Pi
K E DHP DHN-P
ADC
pABA-Glc
Glu
H PPi B
D DHF DHN-P3
Chorismate
* 3
I A
THF THF 2 THF GTP
* 4 Glu
Glu J3 J 2
Glu J1
Folate-Glun Folate-Glun Folate-Glun
5 6
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pABA-Glc
L * 7
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Vacuole
8 9
Folates Pterins
Figure 2
The folate biosynthesis pathway and its compartmentation. Red letters denote enzymes that mediate
biosynthetic steps or ancillary reactions: A, GTP cyclohydrolase I (EC 3.5.4.16); B, dihydroneopterin
(DHN) triphosphate diphosphatase (EC 3.6.1.n4); C, DHN aldolase (EC 4.1.2.25); D,
aminodeoxychorismate synthase (EC 2.6.1.85); E, aminodeoxychorismate lyase (EC 4.1.3.38);
F, 6-hydroxymethyldihydropterin (HMDHP) pyrophosphokinase (EC 2.7.6.3); G, dihydropteroate (DHP)
synthase (EC 2.5.1.15); H, dihydrofolate (DHF) synthase (EC 6.3.2.12); I, DHF reductase (EC 1.5.1.3);
J1–J3, isoforms of folylpolyglutamate synthase (EC 6.3.2.17); K, UDP-glucose–p-aminobenzoate ( pABA)
glucosyltransferase; L, γ-glutamyl hydrolase (EC 3.4.19.9). Circled numbers designate known or inferred
transporters; only those marked with an asterisk (transporters 3, 4, and 7) are cloned. Abbreviations: ADC,
aminodeoxychorismate; DHM, dihydromonapterin; Glu, glutamate; Glun , polyglutamate; -P, phosphate;
P2 , diphosphate; P3 , triphosphate; pABA-Glc, p-aminobenzoate β-D-glucopyranosyl ester; THF,
tetrahydrofolate.
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The HMDHP and pABA precursors are as- certainly not FPGS or the ATP required for the
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sembled into THF in the mitochondrion FPGS reaction (27, 74). Vacuoles presumably
(Figure 2, steps F–I). HMDHP is first ac- import polyglutamates, as discussed below.
tivated by pyrophosphorylation, then coupled The folate polyglutamate tail is not a static
to pABA to yield dihydropteroate. These re- entity but can be shortened or removed by
actions are respectively catalyzed by HMDHP γ-glutamyl hydrolase (GGH), which can have
pyrophosphokinase and dihydropteroate syn- endo- and exopeptidase activities (2, 14, 67).
thase, which are two domains of a single bifunc- GGH is located in vacuoles (Figure 2, step
tional protein in plants (57, 77). Aside from the L) (2, 67). As mentioned above, vacuoles also
canonical, mitochondrially targeted HMDHP contain folate polyglutamates, and vacuolar
pyrophosphokinase–dihydropteroate synthase, GGH activity is sufficiently high to hydrolyze
Arabidopsis has a cytosolic form (86), but this these polyglutamates within minutes (67). That
is expressed only in developing seeds and salt- polyglutamates survive in the vacuole is there-
stressed seedlings and appears to have no coun- fore a mystery. Possible explanations include
terparts in other higher plants. Next, DHF the presence of a potent GGH inhibitor, folate-
synthase couples dihydropteroate to glutamate binding proteins that protect polyglutamates
to yield DHF (74). DHF synthase is unusual from hydrolysis, the partitioning of GGH and
among plant folate synthesis enzymes in that its folate polyglutamates into distinct vacuolar
function has been confirmed by mutant stud- subpopulations, or perhaps intravacuolar com-
ies; disruption of the Arabidopsis gene encod- partmentation similar to that demonstrated for
ing DHF synthase (At5g41480, GLA1) results anthocyanins (54). Although it is not known
in folate deficiency and is embryo lethal (42). how GGH activity is restrained, this activity
Finally, DHF is reduced to THF by DHF re- plays a role in governing polyglutamyl tail
ductase, which in plants is fused to thymidylate length in vivo. Thus, in Arabidopsis leaves and
synthase (15, 53, 61). tomato fruit, overexpressing GGH in vacuoles
The polyglutamate tail is added to THF and reduces average folate polyglutamate tail
its C1 -substituted forms, one residue at a time, length (3); total folate content is also reduced,
via the action of folylpolyglutamate synthase which accords with the idea that polyglutamy-
(FPGS) (41, 74). Arabidopsis has three FPGS lation favors folate binding to proteins and
isoforms, and other higher plants appear to have hence stability (89). Conversely, ablating 99%
two or more (57, 94). On the basis of immuno- of GGH activity in Arabidopsis increases both
logical evidence and green fluorescent protein tail length and total folate content (3). Taken
fusion experiments, the three Arabidopsis iso- together, these results suggest that folates con-
forms appear to be specifically targeted to the tinuously enter the vacuole as polyglutamates,
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accumulate there, are eventually hydrolyzed by ADCS transgenes in tomato fruit causes folate
GGH, and exit as monoglutamates (Figure 2). accumulation and increased expression of the
downstream genes dihydroneopterin aldolase,
aminodeoxychorismate lyase, and mitochon-
Regulation of Folate Synthesis drial FPGS (94). The accumulation of dihy-
The levels of folates and their precursors re- droneopterin (or its phosphates) apparently
main within characteristic ranges for different induces the aldolase, and accumulation of amin-
tissues (44, 66), developmental stages (5, 33, odeoxychorismate induces the lyase; FPGS may
44), and genotypes (32, 69, 88), which implies be induced by accumulation of THF or other
(a) that the synthesis pathway is regulated and monoglutamyl folates (94). Notably, despite
(b) that the regulatory mechanisms vary ge- the massive buildup of pterins and pABA asso-
netically. Despite their pivotal importance for ciated with expression of the GCHI and ADCS
biofortification efforts, these mechanisms are
known only in a fragmentary way; a coher- GTP
A
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feedback mechanism that involves a regulatory vulnerable (34, 39, 78). For THF and DHF,
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protein (99) and also by phosphorylation the first pterins formed in the reaction are
(49). Such mechanisms could not have been tetrahydro- and dihydropterin-6-aldehyde, re-
detected by the published studies of plant spectively; further oxidation can convert the
folate synthesis enzymes because all of them tetrahydro to the dihydro form and both to the
used single recombinant proteins from a fully oxidized, aromatic form pterin-6-aldehyde
heterologous host (E. coli ). (Figure 4a) (38, 78, 97). Additional oxida-
tion converts pterin-6-aldehyde to pterin-6-
FOLATE TURNOVER carboxylate and perhaps other end products
(52).
Folate breakdown rates in plants can be high.
Thus, postharvest studies of leaves and fruits,
and studies using folate synthesis inhibitors, Folate Salvage Processes
point to breakdown rates of ∼10% per day
Like other organisms that synthesize folates,
(66, 70, 83, 88). For comparison, mammalian
plants can recycle the pterin and pABA-Glu
whole-body folate breakdown rates are nor-
cleavage products back to folates (Figure 4b).
mally only ∼0.5% per day (35). As steep
The recycling of pABA-Glu moieties appears to
postharvest declines in folate levels are of obvi-
be straightforward (66). First, the polyglutamyl
ous nutritional significance, understanding the
tail, if present, is removed by GGH, for which
processes involved in folate breakdown and in
pABA-Glu polyglutamates are good substrates
salvage of the breakdown products is as practi-
(2, 67). That GGH is exclusively vacuolar
cally important as understanding de novo folate
implies the existence of a tonoplast transport
biosynthesis.
system for pABA-Glu polyglutamates (which
could be the same as that for folate polyg-
Folate Breakdown lutamates) (Figure 2). Second, pABA-Glu is
As stated in the Introduction, most natural hydrolyzed to yield pABA and glutamate, which
folates are inherently sensitive at physiologi- can then be reused for folate synthesis. An
cal pH to oxidative or photooxidative scission enzyme activity catalyzing this step, pABA-Glu
of the C9–N10 bond, which yields a pterin hydrolase, appears to be ubiquitous in plants, to
plus p-aminobenzoylglutamate ( pABA-Glu) or be predominantly vacuolar or cytosolic, and to
its polyglutamyl forms (Figure 4) (34). Such exist as various isoforms (with native molecular
nonenzymatic cleavage is thought to be the masses of 90, 200, and 360 kDa in Arabidopsis)
main route of folate breakdown in all organisms (12). Partial purification and characterization
(89). However, evidence on this point is lacking of the 200-kDa species from Arabidopsis roots
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showed it to be unstable and probably a in vivo (12). Another possible way to recycle
metalloenzyme (12). The corresponding gene pABA-Glu—direct reincorporation into DHF
has not been cloned, although seven potential via the action of dihydropteroate synthase—was
candidates were tested and ruled out (12). The excluded by a kinetic study of this enzyme (66).
partially purified Arabidopsis root activity also Salvage of the pterin cleavage product is
releases glutamate from folic acid, raising the less well understood, but certain points are
possibility of enzymatic folate degradation fairly clear. First, dihydropterin-6-aldehyde
can be salvaged in vivo by reduction of its
side chain to give the folate synthesis inter-
a THF γ-Glu tail
mediate HMDHP (Figure 4b) (66). In pea
Pterin pABA-Glu leaves, this reaction appears (a) to take place
O H 9 10
O COOH predominantly in the cytosol and (b) to be
N CH2 N C N CH catalyzed by multiple NADPH-dependent
HN H H H
H (CH2)2 COOH pterin aldehyde reductase (PTAR) isoforms
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At1g10310 knockout plants have only a subtle compound can undergo rapid cleavage at
reduction in total PTAR activity (62) but acidic pH to pABA-Glu and a methylated
display an altered fatty acid desaturation pterin (55). Presumably the levels of ascorbate,
phenotype that may be related to the aliphatic glutathione, and other reductants are sufficient
aldehyde reductase activity of At1g10310 (1). in plants to keep 5-methyl-5,6-DHF to a
The combined activity of PTAR isoforms is rel- minimum, but its cleavage may nonetheless
atively high. For instance, in pea leaves, PTAR represent a secondary pathway for nonenzy-
activities are at least 13- to 1,500-fold higher matic folate degradation. In any case, it is not
than those of de novo folate synthesis enzymes known how pterin moieties produced by ox-
(62). This high PTAR activity may favor rapid idative cleavage of 5-methyl- and, presumably,
conversion of dihydropterin-6-aldehyde to 5-formyltetrahydrofolate are metabolized.
HMDHP, thereby minimizing the tendency These pterins presumably still carry a C1
for further oxidation to pterin-6-aldehyde and substituent at the 5-position; in principle, this
pterin-6-carboxylate (Figure 4a). C1 unit might be enzymatically removed, or
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
Second, if PTAR activity fails to intercept removal might not be a necessary precondi-
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other eight include a pterin transporter. is uncertain, given that ablation of At-
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However, complementation tests with FOLT1 affects neither growth, nor leaf
five of these eight proteins in a Leishma- or chloroplast folate content (8). This
nia BT1 knockout strain yielded negative lack of a mutant phenotype may re-
results (26). flect redundancy of function with the
2. Mitochondrial export of THF and/or second chloroplastic folate transporter,
other folates. Because THF is made in At2g32040 (45). At2g32040 and its Syne-
mitochondria, and mitochondria can chocystis ortholog Slr0642 belong to the
convert THF to most of its C1 derivatives FBT family. When expressed in E. coli,
(36), THF and/or C1 folates must be At2g32040 and Synechocystis Slr0642 en-
exported from mitochondria to account able uptake of monoglutamyl folates and
for the folates found elsewhere in the folate analogs (45), whereas other Ara-
cell. Mitochondria can almost certainly bidopsis FBT family proteins do not (26).
also import folates, because supplied Ablation of At2g32040 increases chloro-
5-formyltetrahydrofolate restores folate- plast folate content and reduces the pro-
dependent C1 fluxes in Arabidopsis when portion of 5-methyltetrahydrofolate (45),
folate synthesis is blocked (70); this which provides evidence for a folate
effect requires 5-formyltetrahydrofolate transport role in planta, although growth
to access the mitochondrial enzyme 5- is unaffected. A mutational analysis of
formyltetrahydrofolate cycloligase (79). the Slr0642 protein identified 22 residues
In view of its centrality, mitochondrial fo- essential to folate transport activity, of
late transport is one of the most important which seven are conserved in all known
folate-related processes to understand in FBT family folate transporters (26). The
plants, but nothing is known about it in majority of the eight Arabidopsis FBT pro-
terms of either biochemical activity or teins other than At2g32040 lack at least
genes. In contrast, the mammalian mito- one of these residues, which suggests that
chondrial folate transporter (MFT) has they may transport other substrates. In-
been cloned and extensively characterized terestingly, the FBT family includes a
(68); it is a member of the mitochondrial member, from Leishmania, that is a spe-
carrier family. However, the closest ho- cific, high-affinity S-adenosylmethionine
molog of MFT in Arabidopsis (At5g66380, transporter (22).
AtFOLT1), although it demonstrates fo- 5. Vacuolar pABA glucose ester import.
late transport activity in two heterologous It is necessary to invoke a tonoplast
systems, is targeted to the chloroplast transporter for pABA glucose ester
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because the ester is made in the cytosol accumulation of, methotrexate (73),
but is almost entirely located in vac- indicating that this protein functions in
uoles and, being hydrophilic, almost cer- planta, at least in folate analog transport.
tainly cannot diffuse readily across mem- Whether it plays a significant role in
branes (24, 72). No biochemical evidence folate transport is less certain. First, the
on the transport process is available, and Km values for folate for both AtMRP1
no gene has been cloned. In both these and the beet vacuole system are very high
respects pABA glucose transport is not (0.19 mM) compared with the intracel-
alone: Although many glucose conju- lular concentrations of free (i.e., non–
gates of natural products undergo carrier- protein bound) folate monoglutamates,
mediated uptake into the vacuole, little which are probably submicromolar (73).
is known about the mechanisms or genes Second, like other MRPs, AtMRP1 trans-
(17). ports glutathione conjugates and thus is
6. Vacuolar folate polyglutamate import. not folate specific (73). Third, ablation of
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PP62CH04-Hanson
ARI
+ Arabidopsis ADCS
2007 Rice Grain + Arabidopsis GCHI None Up-to-29-fold increase in pterins 85
+ Arabidopsis ADCS Up to –6-fold Up-to-89-fold increase in pABA
+ Arabidopsis GCHI/ADCS Up to +100-fold Average 4-fold increase in pterins and
25-fold increase in pABA
2008 Rice Leaves + Wheat HPPK/DHPS +75% Precursors not analyzed 29
Grain +40%
2009 Maize Grain + E. coli GCHI +2-fold Precursors not analyzed; β-carotene and 60
ascorbate pathways also engineered
2009 Lettuce Leaves + Chicken GCHIa +2- to +9-fold Precursors not analyzed 64
2010 Arabidopsis Leaves + AtGGH2 –39% Polyglutamate tail length decreased 3
2010 Tomato Fruit + LeGGH2 –46% Polyglutamate tail length decreased 3
2010 Arabidopsis Leaves + GGH RNAi +30% Polyglutamate tail length increased 3
a
Codon usage modified to improve plant expression.
b
Abbreviations: 5-FCL, 5-formyltetrahydrofolate cycloligase; ADCS, aminodeoxychorismate synthase; GCHI, GTP cyclohydrolase I; HHPK/DHPS, 6-hydroxymethyldihydropterin
pyrophosphokinase–dihydropteroate synthase; pABA, p-aminobenzoate; RNAi, RNA interference.
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www.annualreviews.org • Folate Synthesis and Metabolism
4.13
PP62CH04-Hanson ARI 22 January 2011 11:19
of ADCS alone is useless at best. The pABA relied on simple one- or two-gene strategies,
accumulations caused by ADCS transgenes are and almost all have overexpressed enzymes at
of less potential concern than pterin accumu- the head of the pathway, which drives flux down
lations because pABA is among the compounds the pathway by increasing precursor supply.
that are generally recognized as safe (GRAS) This type of “push” strategy inevitably entails
in terms of regulatory status (21, 85). a buildup of precursors, which is undesirable,
The only synthesis engineering study that as mentioned above. More desirable would be
did not manipulate GCHI or ADCS ex- a “pull” strategy in which the folate end prod-
pressed a wheat HMDHP pyrophosphokinase– uct is sequestered in vacuoles, thereby relieving
dihydropteroate synthase gene in rice by using the (little understood) feedback controls over
a constitutive promoter (29). Small (≤75%) in- pathway activity and so enhancing flux without
creases in leaf and grain folate content were precursor accumulation. Although we do not
reported, which suggests that one or another yet know enough about vacuolar folate trans-
of the activities of this bifunctional enzyme ex- port and storage to implement such a strategy,
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
erts significant control of flux through the fo- its intrinsic advantages provide a sound ratio-
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late synthesis pathway, a possibility also raised nale for research to make it possible.
by analysis of engineered tomato fruit (21). The second observation concerns serendip-
A rational engineering strategy might there- ity and arises from a retrospective study of folate
fore be to insert HMDHP pyrophosphokinase– pathway gene expression in engineered tomato
dihydropteroate synthase into plants that ex- fruit (mentioned in the section entitled Regu-
press both GCHI and ADCS transgenes. lation of Folate Synthesis) (94). Basically, a cer-
tain amount of luck was involved in the success
of the two-gene (GCHI-plus-ADCS) strategy
Engineering of Polyglutamylation in tomato fruit: Overexpression of these genes
The generally accepted hypothesis that polyg- induced expression of three downstream path-
lutamylation indirectly stabilizes folates by way genes, which presumably enhanced path-
promoting enzyme binding (89) predicts that way flux capacity. Such feedforward effects are
folate levels can be (a) reduced by shorten- neither predictable nor understood and may not
ing polyglutamyl tails and (b) increased by occur in other systems, meaning that the GCHI-
lengthening them. Studies in which GGH plus-ADCS strategy may not necessarily work
was over- or underexpressed in Arabidopsis and as well as in tomato fruit. That it did so in rice
tomato (3) support both predictions (Table 1). (85) is, however, an encouraging sign.
Thus, threefold overexpression of GGH The third observation is that in microorgan-
reduces average tail length and cuts folate isms there exist many variants of the canonical
content by ∼40%, whereas reducing GGH biosynthesis pathway that include alternatives
activity by 99% increases tail length and raises to almost all its enzymes (18). For instance,
folate content by 30%. Because the folates that certain prokaryotes have a radically different
accumulate in engineered tomato fruit and type of GCHI (23), whereas others have a novel
rice grains are largely unglutamylated (21, 85), enzyme that replaces both dihydroneopterin
another rational engineering strategy might be triphosphate diphosphatase and dihydro-
to increase polyglutamylation by suppressing neopterin aldolase (Figure 2, steps B, C) (71).
GGH activity in these systems. Such evolutionary novelties expand the parts
list for engineering projects and in principle
enable construction of hybrid folate synthesis
Observations on Biofortification routes not found in nature. These potential
Four further observations may be made about alternative routes could have the advantage of
folate engineering and biofortification. The escaping endogenous constraints on pathway
first is that all engineering studies to date have flux (whatever those constraints may be).
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PP62CH04-Hanson ARI 22 January 2011 11:19
Finally, conventional breeding based on nat- Other frontiers are at least implicit in the
ural variation may be a good alternative to foregoing sections on biosynthesis regulation,
metabolic engineering and such breeding could turnover, and transport but bear reempha-
be informed by the outcomes of engineering sizing. Regarding biosynthesis regulation, the
experiments (9). Surveys of tomato (9), potato general lack of biochemical studies of enzymes
(32), wheat (69), and strawberry (88) found isolated from plants (as opposed to recombinant
roughly twofold ranges in folate content among proteins) means that if phosphorylation or reg-
cultivars or breeding lines. Such natural varia- ulatory proteins were involved, we would prob-
tion, if heritable, could form the basis for breed- ably not know. Similarly, at the gene level, if fo-
ing programs. Given the engineering evidence late regulons exist, published studies would not
that both pterin and pABA supplies limit fo- necessarily have detected them. Future tran-
late synthesis (Table 1), such programs might scriptomics studies have the potential to do so.
profitably analyze precursors as well as folates For turnover, there are fascinating hints
in parents and progenies. from postharvest and inhibitor studies that fo-
Annu. Rev. Plant Biol. 2011.62. Downloaded from www.annualreviews.org
SUMMARY POINTS
1. Enzymes for each step of the folate synthesis and polyglutamylation pathways have been
cloned and partially characterized as recombinant proteins. The subcellular compart-
mentation of the pathway is broadly understood, as is that of folates themselves.
2. Certain mechanisms that regulate folate biosynthesis at the enzyme and gene levels have
been identified, and engineering studies have identified enzymes that exert significant
control over flux through the biosynthetic pathway.
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PP62CH04-Hanson ARI 22 January 2011 11:19
3. There is evidence that plants exhibit relatively high folate breakdown rates and can salvage
the breakdown products for reuse in folate synthesis. Salvage enzyme activities have been
identified, and one salvage enzyme has been cloned.
4. Three plant folate transporters—two chloroplastic and one vacuolar—have been cloned.
Enzyme and folate compartmentation data point to the existence of at least five other
folate transport systems in plant cells.
5. Metabolic engineering efforts that overexpressed two folate synthesis genes in combi-
nation have increased folate levels by up to 25-fold in tomato fruit and 100-fold in rice
grains. Lesser increases have been obtained in these and other species through the over-
expression of single genes.
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FUTURE ISSUES
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1. Most of the steps in plant folate synthesis lack genetic confirmation, so it is not clear
whether the known enzymes are the only, or even the major, significant ones in vivo. It
is also unclear whether the pathway is active in all cells or whether some cells depend on
folate import.
2. Knowledge of folate biosynthesis regulation is fragmentary, and no coherent overall
picture has emerged. Comparative biochemistry suggests the possibility of enzyme-level
regulation by phosphorylation and specific regulatory proteins, but no study carried out
so far could have detected such mechanisms.
3. The high rates of folate breakdown in plants suggest that this process may have enzyme-
mediated as well as purely chemical components, but this possibility remains wholly
unexplored. Most folate salvage enzymes remain to be cloned and characterized.
4. The crucial transporters that export folates from their site of synthesis in mitochondria,
that import polyglutamyl folates into vacuoles, and that import folates into the cell have,
for the most part, not been biochemically characterized, and none of them have been
cloned.
5. The folate engineering strategies used to date have resulted in buildup of precursors to
high, perhaps undesirable, levels and may have succeeded in part due to luck. Alternative
rational strategies based on increasing the flux capacity of later as well as early pathway
steps, or on manipulating vacuolar sequestration of folates, have not yet been explored,
in part due to lack of basic knowledge.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
Folate metabolism research in the authors’ laboratories has been funded by the U.S. National
Science Foundation (current grant MCB-0839926). We thank Linda Jeanguenin, Océane Frelin,
and Kenneth Ellens for comments on the manuscript.
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