Folate biosynthesis_hanson

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PP62CH04-Hanson ARI 22 January 2011 11:19

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Folate Biosynthesis, Turnover,and Transport in Plants

Andrew D. Hanson1 and Jesse F. Gregory III2

1Horticultural Sciences Department and 2Food Science and Human Nutrition Department,University of Florida, Gainesville, Florida 32611; email: [email protected], [email protected]

Annu. Rev. Plant Biol. 2011. 62:4.14.21

The Annual Review of Plant Biology is online atplant.annualreviews.org

This articles doi:10.1146/annurev-arplant-042110-103819

Copyright c 2011 by Annual Reviews.All rights reserved

1543-5008/11/0602-0001$20.00

Keywords

biofortification, breakdown, compartmentation, engineering, salvage

Abstract

Folates are essential cofactors for one-carbon transfer reactions and are

needed in the diets of humans and animals. Because plants are major

sources of dietary folate, plant folate biochemistry has long been of in-

terest but progressed slowly until the genome era. Since then, genome-

enabled approaches have brought rapid advances: We now know

(a) all the plant folate synthesis genes and some genes of folate turnover

and transport, (b) certain mechanisms governing folate synthesis, and

(c) the subcellular locations of folate synthesis enzymes and of folates

themselves. Some of this knowledge has been applied, simply and suc-

cessfully, to engineer folate-enriched food crops (i.e., biofortification).

Much remains to be discovered about folates, however, particularly inrelation to homeostasis, catabolism, membrane transport, and vacuolar

storage. Understanding these processes, which will require both bio-

chemical and -omics research, should lead to improved biofortification

strategies based on transgenic or conventional approaches.

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THF:tetrahydrofolate

C1: one-carbon

Biofortification: thebreeding of crops toincrease theirnutritional value byusing conventionalcrossing and selectionor genetic engineering

Pterin: a heterocycliccompound containinga pyrazine ring fused toa pyrimidine ring thathas a carbonyl oxygenand an amino group

pABA:p-aminobenzoate

DHF: dihydrofolate

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 4.2

FOLATE BIOSYNTHESIS . . . . . . . . . . 4.3Pterin Synthesis . . . . . . . . . . . . . . . . . . . 4.3

p-Aminobenzoate Synthesis. . . . . . . . . 4.4

Folate Assembly, Polyglutamylation,

and Deglutamylation. . . . . . . . . . . . 4.5

Regulation of Folate Synthesis. . . . . . 4.6

FOLATE TURNOVER . . . . . . . . . . . . . . 4.7

Folate Breakdown . . . . . . . . . . . . . . . . . . 4.7

Folate Salvage Processes . . . . . . . . . . . 4.7

Transport of Folates

and Precursors. . . . . . . . . . . . . . . . . . 4.9

Cloning Plant Folate Transporters

by Homology . . . . . . . . . . . . . . . . . . . 4.12

ENGINEERING FOLATE

L E V E L S . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . 1 2

Engineering of Biosynthesis . . . . . . . . 4.12

Engineering of Polyglutamylation . . 4.14

Observations on Biofortification . . . . 4.14

FOLATE FRONTIERS . . . . . . . . . . . . . . 4.15

INTRODUCTION

Tetrahydrofolate (THF)is an essential cofactor

in almost all forms of life, in which it acts as a

carrier for one-carbon (C1) units in enzymatic

reactions that form amino acids (methionine,glycine, and serine), purines, thymidylate, pan-

tothenate, and formylmethionyltransfer RNA

(14, 38). THF and its C1-substituted deriva-

tives are collectively termed folates (vitamin

B9). Humans and animals are unable to make

folates de novo and hence depend on dietary

sources, especially plants (11, 83). The health

impacts of folate deficiency in humans can be

severe; they include anemia, spina bifida and

other birth defects, and a higher risk of car-

diovascular disease and certain cancers (11, 87).

Because most plant foods are rather low in fo-

lates and folates are lost during processing andcooking, dietary folatedeficiencycan occur eas-

ilyand is widespread in poorer countries as well

as in some populations within richer ones (11,

83). Folate deficiency is consequently a signifi-

cant worldwide public health problem.

The prevalence of folate deficiency has led

theUnited States (since1998) andsubsequently

many other Western countries to mandate fo-

late fortificationthe addition to cereal grainproducts of chemically synthesized folic acid,

which is metabolized to THF (92). An alterna-

tive approach is dietary supplementation with

folic acid, specifically vitamin pills. However,

both fortification and supplementation are dif-

ficult to implementin poorercountries andmay

have inherent drawbacks related to the fact that

folic acid is an unnatural compound (11, 43,

92). Over the past decade, these concerns have

spurred research on plant folate biosynthesis

that is directed toward metabolic engineering

of natural folate content (biofortification). This

new research direction has reinforced the driveto understand plant folate synthesis because of

its fundamental importance in metabolism.

Given that recent research has focused

mainly on folate biosynthesis and its engineer-

ing, most progress has been in these areas,

and reviews have given various aspects of this

progress pride of place (9, 11, 76, 82, 96). In

covering advances in plant folate biosynthesis

and engineering, this review therefore empha-

sizes what is still not known and does likewise

forthe much lessexploredareas of homeostasis,

catabolism, transport, and storage. Through-

outthis review, information from studies of mi-crobes and animals is used to supplement that

available from studies of plants.

THF is a tripartite molecule composed of

pterin, p-aminobenzoate (pABA), and gluta-

mate moieties (Figure 1). The pterin ring of

folate exists naturally in dihydro or tetrahydro

form; only the latter has cofactor activity. The

ring is fully oxidized in folic acid, whichas

noted aboveis not a natural folate, although

it can be reduced via dihydrofolate (DHF) to

THF. C1 units at various levels of oxidation

(formyl, methylene, methyl) can be enzymati-

callyattachedtotheN5and/orN10positionsofTHF (Figure 1); the resulting C1-substituted

folates are enzymatically interconvertible and

serve as C1 donors for various reactions. A key

characteristic of THF, most of its C1 forms,

and DHF is susceptibility to spontaneous

4.2 Hanson Gregory


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Polyglutamyl tail: ashort chain of-linked

glutamate residuesadded enzymatically tothe glutamate moietyof folates

GCHI: GTPcyclohydrolase I

oxidative or photooxidative cleavage of the C9

N10 bond that links the pterin and pABA moi-

eties. This inherent lability underlies the need

of humans and animals for a continual supplyof folate; what is degraded must be replaced, or

deficiency ensues.

A short, -linked chain of additional

glutamate residues (up to approximately six)

is typically attached to the first glutamate

(Figure 1). This polyglutamyl tail is important

to folate function because folate-dependent en-

zymesgenerally prefer polyglutamates, whereas

folate transporters prefer monoglutamyl forms

(89). The tail thus affects both cofactor activity

and membrane transport. Moreover, because

the tail promotes enzyme binding and folates

are far more stable to oxidative cleavage whenbound than when free, polyglutamylation can

indirectly enhance folate stability (89).

FOLATE BIOSYNTHESIS

Folates are present throughout plant cellsin

the mitochondria, plastids, cytosol, and vac-

uoles (3, 13, 30, 61, 67)but are synthesized

only in mitochondria. The pterin and pABA

precursors are made in the cytosol and plastids,

respectively (Figure 2).

Pterin Synthesis

Pterin synthesis (Figure 2, steps AC) be-

gins with the conversion of GTP to dihydro-

neopterin triphosphate, which is mediated by

GTP cyclohydrolase I (GCHI). The plant en-

zyme has an unusual structure. In other organ-

isms, it is a homodecamerof26-kDasubunits,

whereas in plantsGCHI is a homodimerof sub-

units with two tandem domains, each of which

is similar to a canonical GCHI monomer (5,

56). Given that neither domain has a full set of

the residues involved in substrate binding and

catalysis in other GCHIs, it is not clear howplant GCHI functions catalytically (5). Plant

GCHI is thus an interesting target for future

three-dimensional structure-mechanism stud-

ies. Further reasons to pursue such studies are

that GCHI is the committing enzyme of pterin

N

HNNH

5

NH

H2N

O

CH2

H

H

HC

10NH

CH2CH

COOHO

CH2 COOHNH

Tetrahydropterin p-Aminobenzoate Glutamate

C1 unit

N5CH3

OHCN10

N5CHN10

N5CH2N10

N5CHO

Name

5-methyl-THF

10-formyl-THF

5,10-methenyl-THF

5,10-methylene-THF

5-formyl-THF

p-Aminobenzoylglutamate

N

HNN

NH

H2N

O

CH2

H

H

Dihydropterin moietyof dihydrofolate

THF

a

b c

9

Figure 1

Structure of folates. (a) Chemical structure of tetrahydrofolate (THF),monoglutamyl form. The red arrowhead marks the oxidatively labile C9N10bond. A polyglutamyl tail can be attached via the -carboxyl group of theglutamate moiety. (b) The 7,8-dihydropterin moiety of dihydrofolate. (c) The

various one-carbon (C1) substituents of THF.

synthesis in plants and that, atypically, it shows

substrate inhibition by GTP (5). Plant GCHI

is apparently cytosolic (5, 56).

The dihydroneopterin triphosphate prod-

uct of GCHI is then dephosphorylated to di-

hydroneopterin in two steps. The first stepin plants, as in bacteria, is the removal of

pyrophosphate, which yields dihydroneopterin

monophosphate (46). An Arabidopsis enzyme

from the large Nudix family catalyzes this re-

action in vitro (46), but the same protein may

have other functions (65), and its in vivo role

in folate synthesis awaits genetic confirmation.

As with GCHI, the Nudix hydrolase appears

to be cytosolic (46). Hydrolysis of dihydro-

neopterinmonophosphateto dihydroneopterin

maybe carriedout by a nonspecific phosphatase

in plants, as in Escherichia coli (91), but there is

as yet no biochemical or genetic evidence forthis hypothesis for plants, so a specific enzyme

remains a possibility.

The side chain of dihydroneopterin is then

cleaved to 6-hydroxymethyldihydropterin

(HMDHP) and glycolaldehyde by

www.annualreviews.org Folate Synthesis and Metabolism 4.3


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ADC

pABA

Chorismate

pABA

pABA-Glc

pABA-Glc

THF

DHF

GTP

DHN-P3

DHN-P

DHNDHM

HMDHP

Pi

PPi

HMDHP

HMDHP-P2

DHPGlu

Glu

Folate-Glun

THFTHFGlu

Folate-Glun

Glu

Folate-Glun

pABA

Folate-Glun

Folates

Folates

[Glycosides]

Folates

A

B

C

D

E

F

G

H

I

J1J2J3

L

1

23

4

5 6

7

8

K

*

*

*

Pterins

Pterins

9

Mitochondrion

Plastid

Vacuole

Figure 2

The folate biosynthesis pathway and its compartmentation. Red letters denote enzymes that mediatebiosynthetic 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);J1J3, isoforms of folylpolyglutamate synthase (EC 6.3.2.17); K, UDP-glucosep-aminobenzoate (pABA)glucosyltransferase; L, -glutamyl hydrolase (EC 3.4.19.9). Circled numbers designate known or inferredtransporters; 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-D-glucopyranosyl ester; THF,tetrahydrofolate.

ADCS: aminodeoxy-chorismatesynthase

dihydroneopterin aldolase; this enzyme

also mediates the epimerization of dihy-

droneopterin to dihydromonapterin, which

it likewise cleaves to yield HMDHP (31).Arabidopsis and other plants have small, di-

verged dihydroneopterin aldolase families;

their members lack obvious targeting signals

and therefore are presumably cytosolic (31).

Dihydroneopterin and dihydromonapterin can

be metabolized to -D-glycosides, at least intomato fruit engineered to overproduce pterins

(20). Neither the sugar moiety nor the glyco-

syltransferase(s) involved have been identified,

nor is it known whether the glycosides serve as

a mobilizable reserve of pterins.

p-Aminobenzoate Synthesis

pABA is synthesized from chorismate, theprod-

uct of the shikimate pathway, in two steps

(Figure 2, steps D, E). Both steps are localized

in plastids, as is the shikimate pathway (6, 7).

First, chorismate is converted to aminodeoxy-

chorismate by aminodeoxychorismate synthase

(ADCS), a bipartite protein with tandem do-

mains that are homologous to the PabA and

PabB subunits ofE. coliADCS (6, 57). Second,

aminodeoxychorismate lyase converts amin-

odeoxychorismate to pABA (7). pABA can be

esterified to glucosein a reversiblereaction me-

diated by a cytosolic UDP-glucosyltransferase

4.4 Hanson Gregory


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FPGS:

folylpolyglutamatesynthase

GGH: -glutamylhydrolase

(ArabidopsisAt1g05560, UGT75B1) (Figure 2,

step K) (24, 72). The ester is often more abun-

dant than free pABA (72, 101) and is largely

(

88%) sequestered in vacuoles in pea leaves(24). The ester can be reconverted to pABA in

vitro by reversal of the synthesis reaction or via

an esterase activity (72). The relative impor-

tance of these routes in vivo is unknown, and

the protein(s) and gene(s) responsible for the

esterase activity have not been identified.

Folate Assembly, Polyglutamylation,and Deglutamylation

The HMDHP and p ABA precursors are as-

sembled into THF in the mitochondrion

(Figure 2, steps FI). HMDHP is first ac-tivated by pyrophosphorylation, then coupled

to pABA to yield dihydropteroate. These re-

actions are respectively catalyzed by HMDHP

pyrophosphokinase and dihydropteroate syn-

thase, which are twodomains of a single bifunc-

tional protein in plants (57, 77). Aside from the

canonical, mitochondrially targeted HMDHP

pyrophosphokinasedihydropteroate synthase,

Arabidopsis has a cytosolic form (86), but this

is expressed only in developing seeds and salt-

stressed seedlings and appears to have no coun-

terparts in other higher plants. Next, DHF

synthase couples dihydropteroate to glutamateto yield DHF (74). DHF synthase is unusual

among plant folate synthesis enzymes in that its

function has been confirmed by mutant stud-

ies; disruption of the Arabidopsis gene encod-

ing DHF synthase (At5g41480, GLA1) results

in folate deficiency and is embryo lethal (42).

Finally, DHF is reduced to THF by DHF re-

ductase, which in plants is fused to thymidylate

synthase (15, 53, 61).

The polyglutamatetail is added to THF and

its C1-substituted forms, one residue at a time,

via the action of folylpolyglutamate synthase

(FPGS) (41, 74). Arabidopsis has three FPGSisoforms,and otherhigherplants appearto have

two or more (57, 94). On the basis of immuno-

logical evidence and green fluorescent protein

fusion experiments, the three Arabidopsis iso-

forms appear to be specifically targeted to the

cytosol, mitochondria, and plastids (Figure 2,

steps J1J3) (74). However, data from single

and double FPGS knockouts strongly suggest

that one or more FPGS isoforms are targetedto multiple organelles (58). Multiple targeting

could accountfor how, in plants such as tomato,

two FPGS genes suffice (94). In any case, the

presence of FPGS in cytosol, mitochondria, and

plastids is consistentwith thepresenceof polyg-

lutamates in these compartments (13, 61, 67)

and with the generalization (89) that monog-

lutamates are the transported forms of folate.

Vacuoles, however, are exceptions; they con-

tain folate polyglutamates (3, 67) but almost

certainly notFPGS or theATP required forthe

FPGS reaction (27, 74). Vacuoles presumably

import polyglutamates, as discussed below.The folate polyglutamate tail is not a static

entity but can be shortened or removed by-glutamyl hydrolase (GGH), which can have

endo- and exopeptidase activities (2, 14, 67).

GGH is located in vacuoles (Figure 2, step

L) (2, 67). As mentioned above, vacuoles also

contain folate polyglutamates, and vacuolar

GGH activity is sufficiently high to hydrolyze

these polyglutamates within minutes (67). That

polyglutamates survive in the vacuole is there-

fore a mystery. Possible explanations include

thepresence of a potent GGHinhibitor, folate-

binding proteins that protect polyglutamatesfrom hydrolysis, the partitioning of GGH and

folate polyglutamates into distinct vacuolar

subpopulations, or perhaps intravacuolar com-

partmentation similar to that demonstrated for

anthocyanins (54). Although it is not known

how GGH activity is restrained, this activity

plays a role in governing polyglutamyl tail

length in vivo. Thus, in Arabidopsis leaves and

tomato fruit, overexpressing GGH in vacuoles

reduces average folate polyglutamate tail

length (3); total folate content is also reduced,

which accords with the idea that polyglutamy-

lation favors folate binding to proteins andhence stability (89). Conversely, ablating 99%

of GGH activity in Arabidopsis increases both

tail length and total folate content (3). Taken

together, these results suggest that folates con-

tinuously enter the vacuole as polyglutamates,



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accumulate there, are eventually hydrolyzed by

GGH, and exit as monoglutamates (Figure 2).

Regulation of Folate Synthesis

The levels of folates and their precursors re-

main within characteristic ranges for different

tissues (44, 66), developmental stages (5, 33,

44), and genotypes (32, 69, 88), which implies

(a) that the synthesis pathway is regulated and

(b) that the regulatory mechanisms vary ge-

netically. Despite their pivotal importance for

biofortification efforts, these mechanisms are

known only in a fragmentary way; a coher-

ent overall picture has yet to emerge. Present

knowledge is summarized in Figure 3. As in

other biosynthetic pathways, regulatory loopsappear to operate at both enzyme and gene lev-

els. Thus, in vitro studies have demonstrated

strong feedback inhibition of dihydropteroate

synthase by dihydropteroate, DHF, and THF

(59). Dihydropteroate and DHF also inhibit

the pABA synthesis enzyme ADCS (81), but it

seems unlikely that this effect enables feedback

control in vivo because dihydropteroate and

DHF pools are, presumably, mainly mitochon-

drial, whereas ADCS is plastidial. No other

feedback-regulatory properties of plant pABA

or pterin synthesis enzymes have been demon-

strated (5,6, 7, 31). However, forhumanFPGS,high folate substrate concentrations curtail the

formation of long-chain polyglutamates (93),

and the low average polyglutamyl tail lengths

in plants engineered to overproduce folates (21,

85) are consistent with a similar negative effect

of high folate levels on plant FPGS enzymes.

Transcriptomic analyses have provided

evidence for both feedback and feedforward

regulation of the expression of folate pathway

genes. Specifically, blocking folate synthesis in

Arabidopsis cells with the folate analog

methotrexate (a DHF reductase inhibitor)

causes folate depletion and an increase intranscript level of a single folate synthesis gene,

the cytosolic isoform of FPGS (51), which

suggests that folate polyglutamates exercise

feedback control over expression of this gene.

Conversely, overexpression of the GCHI and

ADCS transgenes in tomato fruit causes folate

accumulation and increased expression of the

downstream genes dihydroneopterin aldolase,

aminodeoxychorismate lyase, and mitochon-drial FPGS (94). The accumulation of dihy-

droneopterin (or its phosphates) apparently

induces thealdolase, andaccumulation of amin-

odeoxychorismate induces the lyase;FPGS may

be induced by accumulation of THF or other

monoglutamyl folates (94). Notably, despite

the massive buildup of pterins and pABA asso-

ciated with expression of the GCHI and ADCS

Mitochondrion

Plastid

GTP

DHN-P3

DHN

HMDHP

DHN-P

HMDHP-P2

DHP

DHF

THF

Glu

Folate-Glun

ADC

Chorismate

pABA

Glu +

+

+

THF

Folate-Glun

J2Glu

J1

I

H

G

F

C

A

B

D

E

Figure 3

Potential regulatory loops operating at the enzymeand gene levels in folate biosynthesis. Broken purplelines denote potential enzyme-level feedbackinhibition mechanisms identified by in vitro studies.Broken blue lines denote gene-level feedbackrepression (minus signs) or feedforward activation(plus signs) mechanisms inferred from transcriptomedata. Folate biosynthesis enzymes are shown byletters as in Figure 2. Dihydromonapterin has beenomitted for simplicity. Abbreviations: ADC,aminodeoxychorismate; DHF, dihydrofolate;DHN, dihydroneopterin; DHP, dihydropteroate;Glu, glutamate; Glun, polyglutamate; HMDHP,6-hydroxymethyldihydropterin; P, phosphate;P2, diphosphate; P3, triphosphate; pABA,p-aminobenzoate; THF, tetrahydrofolate.

4.6 Hanson Gregory


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Turnover: the

dynamic balancebetween synthesis anddegradation of abiomolecule

pABA-Glu: p-amino-benzoylglutamate

transgenes, expression of the endogenous

GCHI and ADCS genes is unaffected (94).

Thus, neither gene-level nor enzyme-level

feedback appears to control the committingreactions of pterin and p ABA synthesis, yet

metabolic engineering studies have demon-

strated that these two reactions exert major

control over flux through the folate pathway

(21, 85). Furthermore, folate synthesis in rice

grains is strongly inhibited in lines engineered

to overproduce p ABA (85). Such paradoxical

observations nicely illustrate how little the

regulation of folate biosynthesis is understood.

Lastly, mammalian GCHI is regulated by a

feedback mechanism that involves a regulatory

protein (99) and also by phosphorylation

(49). Such mechanisms could not have beendetected by the published studies of plant

folate synthesis enzymes because all of them

used single recombinant proteins from a

heterologous host (E. coli).

FOLATE TURNOVER

Folate breakdown rates in plants can be high.

Thus, postharvest studies of leaves and fruits,

and studies using folate synthesis inhibitors,

point to breakdown rates of 10% per day

(66, 70, 83, 88). For comparison, mammalian

whole-body folate breakdown rates are nor-

mally only 0.5% per day (35). As steep

postharvest declines in folate levels are of obvi-

ous nutritional significance, understanding the

processes involved in folate breakdown and in

salvage of the breakdown products is as practi-

cally important as understanding de novo folate

biosynthesis.

Folate Breakdown

As stated in the Introduction, most natural

folates are inherently sensitive at physiologi-

cal pH to oxidative or photooxidative scission

of the C9N10 bond, which yields a pterinplus p-aminobenzoylglutamate (pABA-Glu) or

its polyglutamyl forms (Figure 4) (34). Such

nonenzymatic cleavage is thought to be the

mainrouteof folate breakdownin all organisms

(89). However, evidence on this point is lacking

for plants, so enzyme-mediated cleavage can-

not be excluded; an active cleavage process is

suggested by the unusually high folate break-

down rates in plants. Folate-cleaving enzymesfrom microorganisms have been reported (84),

and mammalian ferritin facilitates folate cleav-

age in vitro and in vivo (90). Moreover, the

action of the folate-dependent COG0354 pro-

tein, which participates in synthesis or repair of

certain iron-sulfur clusters, may involve folate

oxidation (95).

Folates vary in susceptibility to cleavage:

THF and DHF are the most vulnerable, and

5- and 10-formyltetrahydrofolate are the least

vulnerable (34, 39, 78). For THF and DHF,

the first pterins formed in the reaction are

tetrahydro- and dihydropterin-6-aldehyde, re-spectively; further oxidation can convert the

tetrahydro to the dihydro form and both to the

fullyoxidized,aromatic formpterin-6-aldehyde

(Figure 4a) (38, 78, 97). Additional oxida-

tion converts pterin-6-aldehyde to pterin-6-

carboxylate and perhaps other end products

(52).

Folate Salvage Processes

Like other organisms that synthesize folates,

plants can recycle the pterin and pABA-Glu

cleavage products back to folates (Figure 4b).The recycling ofpABA-Glu moieties appears to

be straightforward (66). First, the polyglutamyl

tail, if present, is removed by GGH, for which

pABA-Glu polyglutamates are good substrates

(2, 67). That GGH is exclusively vacuolar

implies the existence of a tonoplast transport

system for pABA-Glu polyglutamates (which

could be the same as that for folate polyg-

lutamates) (Figure 2). Second, p ABA-Glu is

hydrolyzed to yieldpABA and glutamate, which

can then be reused for folate synthesis. An

enzyme activity catalyzing this step, pABA-Glu

hydrolase,appears to be ubiquitous in plants, tobe predominantly vacuolar or cytosolic, and to

exist as various isoforms (with native molecular

masses of 90, 200, and 360 kDa in Arabidopsis)

(12). Partial purification and characterization

of the 200-kDa species from Arabidopsis roots



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showed it to be unstable and probably a

metalloenzyme (12). The corresponding gene

has not been cloned, although seven potential

candidates were tested and ruled out (12). Thepartially purified Arabidopsis root activity also

releases glutamate from folic acid, raising the

possibility of enzymatic folate degradation

a

N

HNN

NH

H2N

O

CHO

HH

Dihydropterin-6-aldehyde

N

HNN

NH2N

O

CH2OH

6-Hydroxymethylpterin

N

HNN

NH2N

O

CHO

Pterin-6-aldehyde

N

HNN

NH2N

OCOOH

Pterin-6-carboxylate

C

-Glu tail

N

HNNH

NH

H2N

O

CH29

H

H

H

NH

10NH

O

pABA-Glu

THF

Pterin

N

HNN

NH

H2N

O

CH2OH

HH

6-HMDHP

O

C

CH

COOH

(CH2)2

NH CH

COOH

NH

CH

COOH

COOH

n

C

O

(CH2)2

(CH2)2

bGTP

Chorismate

HMDHPHMDHP-P2

DHP

DHF

THF

Folate-Glun

Glu

pABA

Glu

pABA-Glun DHPAld

pABA-Glu

GGH

PGH

PTAR

PTAR

PTAR

PTAD

in vivo (12). Another possible way to recycle

p ABA-Gludirect reincorporation into DHF

viathe actionof dihydropteroatesynthasewas

excluded by a kinetic study of this enzyme (66).Salvage of the pterin cleavage product is

less well understood, but certain points are

fairly clear. First, dihydropterin-6-aldehyde

can be salvaged in vivo by reduction of its

side chain to give the folate synthesis inter-

mediate HMDHP (Figure 4b) (66). In pea

leaves, this reaction appears (a) to take place

predominantly in the cytosol and (b) to be

catalyzed by multiple NADPH-dependent

pterin aldehyde reductase (PTAR) isoforms

that can reduce both dihydropterin-6-aldehyde

and pterin-6-aldehyde; similar multiple PTAR

isoforms also occur in Arabidopsis seeds (62).One Arabidopsisisoform (At1g10310) has been

cloned; it belongs to the short-chain dehydro-

genase/reductase family and attacks diverse

aromatic and aliphatic aldehydes (62). Like-

wise, all thepea isoforms attack other aldehydes

(62). Dihydropterin-6-aldehyde thus seems not

to be salvaged by a single dedicated enzyme but

rather by a battery of broad-specificity alde-

hyde reductases. In support of this hypothesis,

Figure 4

Folate breakdown and salvage reactions.(a) Structures and relationships of folate breakdownproducts. Arrowheads mark the oxidatively labileC9N10 bond (red), the bond cleaved byp-aminobenzoylglutamate hydrolase (black), andbonds cleaved by-glutamyl hydrolase (GGH;yellow). Dotted arrows show (photo)chemicaloxidations. Solid arrows show enzymatic reactions;red crosses mark those that appear not to occur inplants (although they occur in other organisms).

The folate synthesis intermediate6-hydroxymethyldihydropterin (HMDHP) iscolored blue. (b) Folate salvage reactions (purplearrows) in relation to folate biosynthesis.

Abbreviations: DHF, dihydrofolate;DHP, dihydropteroate; DHPAld, dihydropterin-6-aldehyde; GGH, -glutamyl hydrolase; Glu,glutamate; Glun, polyglutamate; P2, diphosphate;pABA-Glu, p-aminobenzoylglutamate; PGH,p-aminobenzoylglutamate hydrolase; PTAD, pterinaldehyde dehydrogenase; PTAR, pterin aldehydereductase; THF, tetrahydrofolate.

4.8 Hanson Gregory


9/21


At1g10310 knockout plants have only a subtle

reduction in total PTAR activity (62) but

display an altered fatty acid desaturation

phenotype that may be related to the aliphaticaldehyde reductase activity of At1g10310 (1).

The combined activity of PTAR isoforms is rel-

atively high. For instance, in pea leaves, PTAR

activities are at least 13- to 1,500-fold higher

than those of de novo folate synthesis enzymes

(62). This high PTAR activity may favor rapid

conversion of dihydropterin-6-aldehyde to

HMDHP, thereby minimizing the tendency

for further oxidation to pterin-6-aldehyde and

pterin-6-carboxylate (Figure 4a).

Second, if PTAR activity fails to intercept

dihydropterin-6-aldehyde before it is oxidized

to pterin-6-aldehyde, then the pterin moietycan no longer be salvaged and can only be fur-

ther oxidized (63). Such a situation arises be-

cause plants lack the capacity to reduce the

fully oxidized pterin ring to the dihydro state,

a deficit shared with E. coli (63). The lack of

pterin-reducing capacityis surprising inasmuch

as enzymes with this activity occur in other or-

ganisms; pteridine reductase 1 ofLeishmania is

one example (10). Further oxidation of pterin-

6-aldehyde to pterin-6-carboxylate can occur

spontaneously or enzymatically; plant extracts

have both NAD-dependent and -independent

activities (63). Pterin-6-carboxylate seems to bea dead-end product; its relative scarcity in most

plant tissues suggests that folate salvage is nor-

mally efficient (66).

The following points about folate salvage

are still obscure. First, the oxidative cleavage

product of THF is tetrahydropterin-6-

aldehyde (89), and it is not clear how oxidation

to the dihydro form occurs or even whether

it is necessary (given that the tetrahydro form

could conceivably be recycled directly to

THF). Second, due to the resistance of 5-

methyl- and 5- and 10-formyltetrahydrofolates

to oxidative cleavage (55), it is likely that THFand DHF are the primary folates that undergo

nonenzymatic oxidative cleavage. However,

in the case of 5-methyltetrahydrofolate,

for example, facile and reversible oxidation

to 5-methyl-5,6-DHF can occur, and this

compound can undergo rapid cleavage at

acidic pH to p ABA-Glu and a methylated

pterin (55). Presumably the levels of ascorbate,

glutathione, and other reductants are sufficientin plants to keep 5-methyl-5,6-DHF to a

minimum, but its cleavage may nonetheless

represent a secondary pathway for nonenzy-

matic folate degradation. In any case, it is not

known how pterin moieties produced by ox-

idative cleavage of 5-methyl- and, presumably,

5-formyltetrahydrofolate are metabolized.

These pterins presumably still carry a C1substituent at the 5-position; in principle, this

C1 unit might be enzymatically removed, or

removal might not be a necessary precondi-

tion for recycling. Lastly, the predominantly

cytosolic location of PTAR activity seemsanomalous, given that 3050% of the folates

in metabolically active plant cells are in mito-

chondria (28, 44, 61) and that mitochondrial

folates are almost certainly at a high risk

of oxidative cleavage due to the prevalence

of reactive oxygen species in mitochondria

(50). The implication is that pterin cleavage

products cannot be recycled until they are

exported from the mitochondria, which seems

to be at odds with the need for PTAR to

intervene before further oxidation puts the

pterins beyond rescue.

Transport of Folates and Precursors

The compartmentation of the enzymes of

folate biosynthesis and metabolism, and of

folates themselves, means that there must be

substantial transmembrane traffic in folates

and their precursors. Of this traffic, only that in

pABA (a hydrophobic weak acid) appears to be

by simple diffusion (72). On the basis of com-

partmentation data, experimental evidence,

and comparative biochemistry, a minimal set of

nine probable carrier-mediated transport steps

can be defined (Figure 2, steps 19). Thesesteps, and the evidence for them, are as follows.

1. Mitochondrial pterin import. If

HMDHP is synthesized in the cy-

tosol, it must enter mitochondria

to support folate synthesis. Also,



10/21


mitochondria probably export pterins

resulting from folate degradation (see

above). Pterin transport has not been

studied in plant mitochondria, but itseems likely to be carrier mediated given

that the only well-studied pterin uptake

process, in the protistan parasiteLeishma-

nia, involves a specific transporter (BT1)

that belongs to the folate-biopterin

transporter (FBT) family (48). The

Arabidopsis genome encodes nine FBT

family proteins, only one of which has a

known function (in folate transport; see

below). It is therefore possible that the

other eight include a pterin transporter.

However, complementation tests with

five of these eight proteins in a Leishma-nia BT1 knockout strain yielded negative

results (26).

2. Mitochondrial export of THF and/or

other folates. Because THF is made in

mitochondria, and mitochondria can

convert THF to most of its C1 derivatives

(36), THF and/or C1 folates must be

exported from mitochondria to account

for the folates found elsewhere in the

cell. Mitochondria can almost certainly

also import folates, because supplied

5-formyltetrahydrofolate restores folate-

dependent C1 fluxes in Arabidopsis whenfolate synthesis is blocked (70); this

effect requires 5-formyltetrahydrofolate

to access the mitochondrial enzyme 5-

formyltetrahydrofolate cycloligase (79).

In view of its centrality, mitochondrial fo-

latetransportisoneofthemostimportant

folate-related processes to understand in

plants, but nothing is known about it in

terms of either biochemical activity or

genes. In contrast, the mammalian mito-

chondrial folate transporter (MFT) has

beencloned and extensivelycharacterized

(68); it is a member of the mitochondrialcarrier family. However, the closest ho-

mologofMFTinArabidopsis(At5g66380,

AtFOLT1), although it demonstrates fo-

late transport activity in two heterologous

systems, is targeted to the chloroplast

envelope, not to mitochondria (8). Two

other, less close Arabidopsis homologs

of MFT appear to lack folate transport

activity (8).3,4. Plastidial folate import. Two chloro-

plast envelope proteins that can medi-

ate folate transport have been identi-

fied in Arabidopsis. Both are homologs

of known folate transporters. The first,

AtFOLT1 (8), is described above. At-

FOLT1 complements the mft mutation

in Chinese hamster ovary cells and con-

fers folateuptakewhen expressedinE.coli

cells. However, its significance in planta

is uncertain, given that ablation of At-

FOLT1 affects neither growth, nor leaf

or chloroplast folate content (8). Thislack of a mutant phenotype may re-

flect redundancy of function with the

second chloroplastic folate transporter,

At2g32040 (45). At2g32040 and its Syne-

chocystis ortholog Slr0642 belong to the

FBT family. When expressed in E. coli,

At2g32040 and Synechocystis Slr0642 en-

able uptake of monoglutamyl folates and

folate analogs (45), whereas other Ara-

bidopsisFBT family proteins do not (26).

Ablation of At2g32040 increases chloro-

plast folate content and reduces the pro-

portion of 5-methyltetrahydrofolate (45), which provides evidence for a folate

transport role in planta, although growth

is unaffected. A mutational analysis of

the Slr0642 protein identified 22 residues

essential to folate transport activity, of

which seven are conserved in all known

FBT family folate transporters (26). The

majority of theeightArabidopsisFBTpro-

teins other than At2g32040 lack at least

one of these residues, which suggests that

they may transport other substrates. In-

terestingly, the FBT family includes a

member, from Leishmania, that is a spe-cific, high-affinityS-adenosylmethionine

transporter (22).

5. Vacuolar p ABA glucose ester import.

It is necessary to invoke a tonoplast

transporter for p ABA glucose ester

4.10 Hanson Gregory


11/21


because the ester is made in the cytosol

but is almost entirely located in vac-

uoles and, being hydrophilic, almost cer-

tainly cannot diffuse readily across mem-branes (24, 72). No biochemical evidence

on the transport process is available, and

no gene has been cloned. In both these

respects p ABA glucose transport is not

alone: Although many glucose conju-

gates of natural products undergo carrier-

mediated uptake into the vacuole, little

is known about the mechanisms or genes

(17).

6. Vacuolar folate polyglutamate import.

The argument for a tonoplast transport

system for folate polyglutamates is

outlined above (see the section entitledFolate Assembly, Polyglutamylation, and

Deglutamylation); there is no biochem-

ical information on this system, and no

gene is known. Although such a system

would be unusual in that most folate

transporters strongly prefer monoglu-

tamyl folates (25, 45, 89), a precedent

exists in mammalian lysosomes, which,

like plant vacuoles, import polyglu-

tamates and contain GGH (4). The

mammalian polyglutamate transport sys-

tem has unfortunately not been cloned.

7. Vacuolar folate monoglutamate import.Folates and their analogs are substrates

for certain mammalian multidrug

resistanceassociated protein (MRP)

subfamily ATP-binding cassette trans-

porters (100), and there is evidence that

MRP proteins import folates into plant

vacuoles. Thus, the Arabidopsis vacuolar

MRP protein AtMRP1 (At1g30400)

expressed in yeast, and its functional

equivalent(s) in vacuolar membrane vesi-

cles from red beet root, are competent in

the MgATP-dependent transport of folic

acid and methotrexate (73). Polyglutamylfolates are poor substrates, so AtMRP1

is unlikely to correspond to the polyg-

lutamate transporter discussed above.

Ablation of AtMRP1 results in increased

sensitivity to, and impaired vacuolar

accumulation of, methotrexate (73),

indicating that this protein functions in

planta, at least in folate analog transport.

Whether it plays a significant role infolate transport is less certain. First, the

Km values for folate for both AtMRP1

and the beet vacuole system are very high

(0.19 mM) compared with the intracel-

lular concentrations of free (i.e., non

protein bound) folate monoglutamates,

which are probably submicromolar (73).

Second, likeother MRPs, AtMRP1trans-

ports glutathione conjugates and thus is

not folate specific (73). Third, ablation of

AtMRP1 eliminates only approximately

half of vacuolar methotrexate uptake ac-

tivity, which indicates the presence of atleast one other transport system. Finally,

because ATP-binding cassette trans-

porters work in only one direction (75),

AtMRP1 is unlikely to mediate the ex-

port from vacuoles of the monoglutamyl

folate products of GGH action.

8. Cellular folate import. There is strong

evidence that plant cells or tissues can

take up intact folates from experiments

in which supplied folates are metabolized

(70, 80)or reverse theeffects of folatesyn-

thesis inhibitors (16, 51). There is like-

wise direct evidence for uptake of theclose folate analog methotrexate (16, 51,

70), including a 1993 study ofDatura in-

noxia cells that determined a Km value of

66 nM and showed that uptake is pH and

energy dependent (98). The same study

also showed that selection for methotrex-

ateresistanceresultedinanincreaseinthe

Km value for methotrexate uptake. This

pioneering work invites extension to the

gene level via the molecular and genetic

tools now available.

9. Cellular pterin import. There is good

evidence that plant tissues take up andmetabolize supplied pterins (62, 66), as

do E. coli, other bacteria, and yeast (63).

Pterin transport is, however, a gener-

ally neglected research area and has been

studied only in Leishmania (48).



12/21


Cloning Plant Folate Transportersby Homology

The cloning-by-homology approach that

yielded AtFOLT1, At2g32040, and AtMRP1may have ceased to be fruitful. Taking all

organisms together, seven classes of folate

transporters have so far been cloned (26). Five

are from mammals: the mitochondrial trans-

porter, the reduced folate carrier, the intestinal

protoncoupled folate transporter, various

MRPs, and glycosylphosphatidylinisotol-

linked folate receptors, which mediate uptake

by an endocytic mechanism. Of these, only

the mitochondrial and MRP transporters have

reasonably close plant homologs (AtFOLT1

and AtMRP1, respectively), which are known

to transport folates, as described above. Thesixth class is the FBT family, one of whose

plant homologs (At2g32040) also transports

folates. The seventh class, members of the

bacterial energycoupling factor family, do not

have homologs in plants.

ENGINEERING FOLATE LEVELS

As summarized in Table 1, the biosynthesis

pathway and the control of polyglutamylation

have been the targets for almost all plant folate

engineering studies so far. The sole exceptionis an Arabidopsis model study (30) designed to

enhance folate content by blocking metabolism

of 5-formyltetrahydrofolate, as proposed by

Scott et al. (83). This strategy indeed raises

total folate level, but only by twofold, and

leads to slowed growth and delayed flowering.

It is consequently not promising and has not

been pursued. In contrast, several of the other

studies achieved a far-greater-than-twofold

enrichment, and none reported negative effects

on growth or development.

Engineering of Biosynthesis

As is common in pathway engineering studies,

various species, organs, promoters, and ana-

lytical methods have been used by different

groups, which precludes direct comparisons.

Some useful generalizations can nonetheless be

made. Thus, of seven studies that manipulated

the biosynthetic pathway, six inserted genes

coding for GCHI, the first enzyme of pterinsynthesis (Figure 2, step A), alone or together

withADCS, the first enzyme ofpABA synthesis

(Figure 2, step D). In most cases, the GCHI

transgene was from a nonplant source

(E. coli, mouse, or chicken), on the basis of the

reasonable but undocumented premise that

plant GCHI enzymes are subject to feedback

inhibition, which would limit their potential

to increase flux. Expressed alone, the nonplant

transgenes generally led to substantial pterin

overproduction, but so did a plant transgene

( Table 1). It is therefore not clear whether

plant or nonplant GCHIs are preferable, butit can at least be concluded that the former

need not be avoided on principle. Whatever

the case, excessive overproduction of pterins

may be undesirable, not least because their

health effects and potential roles in human

metabolism are unknown (21).

Despite massively boosting pterin levels,

the introduction of a GCHI transgene alone

generally raises folate content only modestly,

typically by approximately twofold (Table 1).

When total p ABA levels (i.e., p ABA plus

its glucose ester) were analyzed in GCHI-

overexpressing tomato fruit (20), severe pABAdepletion was observed, indicating that the

pABA supplylimits further folateaccumulation.

Consistent with this view, adding anArabidopsis

ADCS transgene greatly increases the total

p ABA pool and raises folate content to as

much as 25 times the content in wild-type fruit

(Table 1). Expression of the ADCS transgene

alone had no effect on fruit folate content.

The results of expressing GCHI and ADCS

transgenes separately and together in rice

grains are very similar, except that folate con-

tent is substantially (and unexpectedly) reduced

by expression of ADCS alone (as discussed inthe section entitled Regulation of Folate Syn-

thesis). Taken together, the tomato and rice

data indicate that expression of ADCS in com-

bination with GCHI is far more effective than

expression of GCHI alone, and that expression

4.12 Hanson Gregory


13/21


Ta

ble1

Pro

jec

tsa

iming

tome

tab

oli

ca

llyeng

ineer

folate

con

ten

t

Year

Sp

ec

ies

Organ

(s)teste

d

Gene

(s)ad

de

dora

blate

d

Ch

ange

infolate

Commen

ts

Re

ference

2004

Arabidopsis

Leaves

+

Escherichia

coliGCHI

+2-to+4-fold

1,250-foldincreaseinpterins

40

2004

Tomato

Fruit

+

MouseGC

HIa

+2-fold

Up-to-140-foldincreaseinpterins

20

2005

Arabidopsis

Leaves

5-F

CLablated

+2-fold

Growthslowed20%;floweringdelayed

30

2007

Tomato

Fruit

+

ArabidopsisADCS

None

Up-to-40-foldincreaseinpABA

21

+

MouseGC

HIa/

Upto+25-fold

>20-foldincreasesinpterinsandpABA

+

ArabidopsisADCS

2007

Rice

Grain

+

ArabidopsisGCHI

None

Up-to-29-foldincreaseinpterins

85

+

ArabidopsisADCS

Upto6-fold

Up-to-89-foldincreaseinpABA

+

ArabidopsisGCHI/ADCS

Upto+100-fold

Average4-foldincreaseinpterinsand

25

-foldincreaseinpABA

2008

Rice

Leaves

+

WheatHP

PK/DHPS

+75%

Pre

cursorsnotanalyzed

29

Grain

+40%

2009

Maize

Grain

+

E.

coliGCHI

+2-fold

Pre

cursorsnotanalyzed;-caroteneand

ascorbatepathwaysalsoengineered

60

2009

Lettu

ce

Leaves

+

ChickenG

CHIa

+2-to+9-fold

Pre

cursorsnotanalyzed

64

2010

Arabidopsis

Leaves

+

AtGGH2

39%

Polyglutamatetaillengthdecreased

3

2010

Tomato

Fruit

+

LeGGH2

46%

Polyglutamatetaillengthdecreased

3

2010

Arabidopsis

Leaves

+

GGHRNAi

+30%

Polyglutamatetaillengthincreased

3

aCodonusagemodifiedtoimproveplantexpression.

bAbbreviations:5-FCL,5-formyltetrahydrofolatecycloligase;ADCS,am

inodeoxychorismatesynthase;GCHI,GTPcyclohydrolaseI;HHPK/DHPS,6-hydroxymethyldihydropterin

pyrophosphokinasedihydropteroatesynthase;pABA,p-aminobenzoate;RNA

i,RNAinterference.



14/21


of ADCS alone is useless at best. The pABA

accumulations caused by ADCS transgenes are

of less potential concern than pterin accumu-

lations because pABA is among the compoundsthat are generally recognized as safe (GRAS)

in terms of regulatory status (21, 85).

The only synthesis engineering study that

did not manipulate GCHI or ADCS ex-

pressed a wheat HMDHP pyrophosphokinase

dihydropteroate synthase gene in rice by using

a constitutive promoter (29). Small (75%) in-

creases in leaf and grain folate content were

reported, which suggests that one or another

of the activities of this bifunctional enzyme ex-

erts significant control of flux through the fo-

late synthesis pathway, a possibility also raised

by analysis of engineered tomato fruit (21). A rational engineering strategy might there-

fore be to insertHMDHP pyrophosphokinase

dihydropteroate synthase into plants that ex-

press both GCHI and ADCS transgenes.

Engineering of Polyglutamylation

The generally accepted hypothesis that polyg-

lutamylation indirectly stabilizes folates by

promoting enzyme binding (89) predicts that

folate levels can be (a) reduced by shorten-

ing polyglutamyl tails and (b) increased by

lengthening them. Studies in which GGHwas over- or underexpressed in Arabidopsisand

tomato (3) support both predictions (Table 1).

Thus, threefold overexpression of GGH

reduces average tail length and cuts folate

content by 40%, whereas reducing GGH

activity by 99% increases tail length and raises

folate content by 30%. Because the folates that

accumulate in engineered tomato fruit and

rice grains are largely unglutamylated (21, 85),

another rational engineering strategy might be

to increase polyglutamylation by suppressing

GGH activity in these systems.

Observations on Biofortification

Four further observations may be made about

folate engineering and biofortification. The

first is that all engineering studies to date have

relied on simple one- or two-gene strategies,

and almost all have overexpressed enzymes at

thehead of thepathway, which drivesflux down

the pathway by increasing precursor supply.This type of push strategy inevitably entails

a buildup of precursors, which is undesirable,

as mentioned above. More desirable would be

a pull strategy in which the folate end prod-

uct is sequestered in vacuoles, thereby relieving

the (little understood) feedback controls over

pathway activity and so enhancing flux without

precursor accumulation. Although we do not

yet know enough about vacuolar folate trans-

port and storage to implement such a strategy,

its intrinsic advantages provide a sound ratio-

nale for research to make it possible.

The second observation concerns serendip-ityand arises from a retrospective study of folate

pathway gene expression in engineered tomato

fruit (mentioned in the section entitled Regu-

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

in tomato fruit: Overexpression of these genes

induced expression of three downstream path-

way genes, which presumably enhanced path-

way flux capacity. Such feedforward effects are

neither predictablenor understood and may not

occur in other systems, meaning that theGCHI-

plus-ADCS strategy may not necessarily workas well as in tomato fruit. That it did so in rice

(85) is, however, an encouraging sign.

The third observation is that in microorgan-

isms there exist many variants of the canonical

biosynthesis pathway that include alternatives

to almost all its enzymes (18). For instance,

certain prokaryotes have a radically different

typeof GCHI (23), whereas others havea novel

enzyme that replaces both dihydroneopterin

triphosphate diphosphatase and dihydro-

neopterin aldolase (Figure 2, steps B, C) (71).

Such evolutionary novelties expand the parts

list for engineering projects and in principleenable construction of hybrid folate synthesis

routes not found in nature. These potential

alternative routes could have the advantage of

escaping endogenous constraints on pathway

flux (whatever those constraints may be).

4.14 Hanson Gregory


15/21


Finally, conventional breeding basedon nat-

ural variation may be a good alternative to

metabolic engineering and such breeding could

be informed by the outcomes of engineeringexperiments (9). Surveys of tomato (9), potato

(32), wheat (69), and strawberry (88) found

roughly twofold ranges in folate content among

cultivars or breeding lines. Such natural varia-

tion, if heritable,couldform thebasis forbreed-

ing programs. Given the engineering evidence

that both pterin and p ABA supplies limit fo-

late synthesis (Table 1), such programs might

profitably analyze precursors as well as folates

in parents and progenies.

FOLATE FRONTIERS

The frontiers include all the topics in this re-

view, includingdespite the effort invested in

itthe biosynthesis pathway itself. Specifically,

only one step in this pathway (DHF synthase)

hasbeenvalidatedbymutantstudies(42);allthe

others rest on biochemical data, comparative

genomics, and functional complementation of

microbial mutants. Thus, if alternatives to the

classical folate synthesis steps exist in plants, we

would not know about them. Moreover, it is

not clear whether all cells and organs are au-

tonomous for folate synthesis or whether somedepend on intercellular or interorgan traffic in

folates or their precursors. The presence of a

high-affinity cellular folate uptake system (98)

makes such traffic seem probable, as does ev-

idence that maternal tissues supply folates to

early embryos (42). A first step to address this

issue would bemetabolic profiling of folates and

folate precursors in xylem and phloem sap.

Other frontiers are at least implicit in the

foregoing sections on biosynthesis regulation,

turnover, and transport but bear reempha-

sizing. Regarding biosynthesis regulation, thegeneral lack of biochemical studies of enzymes

isolated fromplants(as opposed to recombinant

proteins) means that if phosphorylation or reg-

ulatory proteins were involved, we would prob-

ably not know. Similarly, at the gene level, if fo-

late regulons exist, published studies would notnecessarily have detected them. Future tran-

scriptomics studies have the potential to do so.

For turnover, there are fascinating hints

from postharvest and inhibitor studies that fo-

late breakdown in plants may be too fast to ex-

plain by spontaneous chemical processes alone,

and other hintsparticularly from compara-tive biochemistrythat folate degradation can

be enzyme mediated. Biochemistry, compara-

tive genomics, transcriptomics, and proteomics

could be applied and integrated to search for

candidate folate-degrading proteins (18, 19, 37,

47). Such work has yet to begin.

Folate transport and vacuolar storage are

perhaps the frontiers with the greatest poten-

tial short-term payoffs in engineering terms.

Few folate transporters have been discovered

in plants, and they are probably the least

interesting ones for engineering. Far more

crucial are the transporters that mediate mito-chondrial folate export, vacuolar folate polyg-

lutamate import, and folate uptakeinto the cell.

Although homology to known folate trans-

portersisnearlyifnotcompletelyminedoutasa

way to identify such transporters, more creative

approaches, including comparative genomics

and other -omics technologies, hold consider-

able potential but, again, have yet to be applied.

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.



16/21


3. There is evidence that plantsexhibit relativelyhigh folatebreakdown rates andcan salvage

the breakdown products for reuse in folate synthesis. Salvageenzyme activities have been

identified, and one salvage enzyme has been cloned.4. Three plant folate transporterstwo chloroplastic and one vacuolarhave 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.

FUTURE ISSUES

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, Oceane Frelin,

and Kenneth Ellens for comments on the manuscript.

4.16 Hanson Gregory


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