A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.

Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.

Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing effects on growth and on activity of the iron-sulfur enzyme MiaB.

The folate-dependent protein YgfZ of Escherichia coli participates in the synthesis and repair of iron-sulfur (Fe-S) clusters; it belongs to a family of enzymes that use folate to capture formaldehyde units. Ablation of ygfZ is known to reduce growth, to increase sensitivity to oxidative stress, and to lower the activities of MiaB and other Fe-S enzymes. It has been reported that the growth phenotype can be suppressed by disrupting the tRNA modification gene mnmE. We first confirmed the latter observation using deletions in a simpler, more defined genetic background. We then showed that deleting mnmE substantially restores MiaB activity in ygfZ deletant cells and that overexpressing MnmE with its partner MnmG exacerbates the growth and MiaB activity phenotypes of the ygfZ deletant. MnmE, with MnmG, normally mediates a folate-dependent transfer of a formaldehyde unit to tRNA, and the MnmEG-mediated effects on the phenotypes of the ΔygfZ mutant apparently require folate, as evidenced by the effect of eliminating all folates by deleting folE. The expression of YgfZ was unaffected by deleting mnmE or overexpressing MnmEG or by folate status. Since formaldehyde transfer is a potential link between MnmEG and YgfZ, we inactivated formaldehyde detoxification by deleting frmA. This deletion had little effect on growth or MiaB activity in the ΔygfZ strain in the presence of formaldehyde, making it unlikely that formaldehyde alone connects the actions of MnmEG and YgfZ. A more plausible explanation is that MnmEG erroneously transfers a folate-bound formaldehyde unit to MiaB and that YgfZ reverses this.

Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism.

COG0354 proteins have been implicated in synthesis or repair of iron/sulfur (Fe/S) clusters in all domains of life, and those of bacteria, animals, and protists have been shown to require a tetrahydrofolate to function. Two COG0354 proteins were identified in Arabidopsis and many other plants, one (At4g12130) related to those of α-proteobacteria and predicted to be mitochondrial, the other (At1g60990) related to those of cyanobacteria and predicted to be plastidial. Grasses and poplar appear to lack the latter. The predicted subcellular locations of the Arabidopsis proteins were validated by in vitro import assays with purified pea organelles and by targeting assays in Arabidopsis and tobacco protoplasts using green fluorescent protein fusions. The At4g12130 protein was shown to be expressed mainly in flowers, siliques, and seeds, whereas the At1g60990 protein was expressed mainly in young leaves. The folate dependence of both Arabidopsis proteins was established by functional complementation of an Escherichia coli COG0354 (ygfZ) deletant; both plant genes restored in vivo activity of the Fe/S enzyme MiaB but restoration was abrogated when folates were eliminated by deleting folP. Insertional inactivation of At4g12130 was embryo lethal; this phenotype was reversed by genetic complementation of the mutant. These data establish that COG0354 proteins have a folate-dependent function in mitochondria and plastids, and that the mitochondrial protein is essential. That plants retain mitochondrial and plastidial COG0354 proteins with distinct phylogenetic origins emphasizes how deeply the extant Fe/S cluster assembly machinery still reflects the ancient endosymbioses that gave rise to plants.

Synergistic use of plant-prokaryote comparative genomics for functional annotations.

BACKGROUND: Identifying functions for all gene products in all sequenced organisms is a central challenge of the post-genomic era. However, at least 30-50% of the proteins encoded by any given genome are of unknown or vaguely known function, and a large number are wrongly annotated. Many of these 'unknown' proteins are common to prokaryotes and plants. We set out to predict and experimentally test the functions of such proteins. Our approach to functional prediction integrates comparative genomics based mainly on microbial genomes with functional genomic data from model microorganisms and post-genomic data from plants. This approach bridges the gap between automated homology-based annotations and the classical gene discovery efforts of experimentalists, and is more powerful than purely computational approaches to identifying gene-function associations. RESULTS: Among Arabidopsis genes, we focused on those (2,325 in total) that (i) are unique or belong to families with no more than three members, (ii) occur in prokaryotes, and (iii) have unknown or poorly known functions. Computer-assisted selection of promising targets for deeper analysis was based on homology-independent characteristics associated in the SEED database with the prokaryotic members of each family. In-depth comparative genomic analysis was performed for 360 top candidate families. From this pool, 78 families were connected to general areas of metabolism and, of these families, specific functional predictions were made for 41. Twenty-one predicted functions have been experimentally tested or are currently under investigation by our group in at least one prokaryotic organism (nine of them have been validated, four invalidated, and eight are in progress). Ten additional predictions have been independently validated by other groups. Discovering the function of very widespread but hitherto enigmatic proteins such as the YrdC or YgfZ families illustrates the power of our approach. CONCLUSIONS: Our approach correctly predicted functions for 19 uncharacterized protein families from plants and prokaryotes; none of these functions had previously been correctly predicted by computational methods. The resulting annotations could be propagated with confidence to over six thousand homologous proteins encoded in over 900 bacterial, archaeal, and eukaryotic genomes currently available in public databases.

Subcellular and tissue localization of NAD kinases from Arabidopsis: compartmentalization of de novo NADP biosynthesis.

The de novo biosynthesis of the triphosphopyridine NADP is catalyzed solely by the ubiquitous NAD kinase family. The Arabidopsis (Arabidopsis thaliana) genome contains two genes encoding NAD+ kinases (NADKs), annotated as NADK1, NADK2, and one gene encoding a NADH kinase, NADK3, the latter isoform preferring NADH as a substrate. Here, we examined the tissue-specific and developmental expression patterns of the three NADKs using transgenic plants stably transformed with NADK promoter::glucuronidase (GUS) reporter gene constructs. We observed distinct spatial and temporal patterns of GUS activity among the NADK::GUS plants. All three NADK::GUS transgenes were expressed in reproductive tissue, whereas NADK1::GUS activity was found mainly in the roots, NADK2::GUS in leaves, and NADK3::GUS was restricted primarily to leaf vasculature and lateral root primordia. We also examined the subcellular distribution of the three NADK isoforms using NADK-green fluorescent protein (GFP) fusion proteins expressed transiently in Arabidopsis suspension-cultured cells. NADK1 and NADK2 were found to be localized to the cytosol and plastid stroma, respectively, consistent with previous work, whereas NADK3 localized to the peroxisomal matrix via a novel type 1 peroxisomal targeting signal. The specific subcellular and tissue distribution profiles among the three NADK isoforms and their possible non-overlapping roles in NADP(H) biosynthesis in plant cells are discussed.

'Unknown' proteins and 'orphan' enzymes: the missing half of the engineering parts list--and how to find it.

Like other forms of engineering, metabolic engineering requires knowledge of the components (the 'parts list') of the target system. Lack of such knowledge impairs both rational engineering design and diagnosis of the reasons for failures; it also poses problems for the related field of metabolic reconstruction, which uses a cell's parts list to recreate its metabolic activities in silico. Despite spectacular progress in genome sequencing, the parts lists for most organisms that we seek to manipulate remain highly incomplete, due to the dual problem of 'unknown' proteins and 'orphan' enzymes. The former are all the proteins deduced from genome sequence that have no known function, and the latter are all the enzymes described in the literature (and often catalogued in the EC database) for which no corresponding gene has been reported. Unknown proteins constitute up to about half of the proteins in prokaryotic genomes, and much more than this in higher plants and animals. Orphan enzymes make up more than a third of the EC database. Attacking the 'missing parts list' problem is accordingly one of the great challenges for post-genomic biology, and a tremendous opportunity to discover new facets of life's machinery. Success will require a co-ordinated community-wide attack, sustained over years. In this attack, comparative genomics is probably the single most effective strategy, for it can reliably predict functions for unknown proteins and genes for orphan enzymes. Furthermore, it is cost-efficient and increasingly straightforward to deploy owing to a proliferation of databases and associated tools.

Identification, molecular cloning and functional characterization of a novel NADH kinase from Arabidopsis thaliana (thale cress).

NADH kinase (NADHK; ATP:NADH 2'-phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg2+ and NAD+, with respective K(m) values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD+, the specificity constant (V(max)/K(m)) for NADH was 100-fold greater than for NAD+. The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PP(i) or poly-P(i) could not substitute as phospho donors. PP(i) acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD+. This study suggests that, in Arabidopsis, NADP+/NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool.

Compartmentation of GABA metabolism raises intriguing questions.

This synopsis covers the compartmentation of γ-aminobutyrate (GABA) metabolism, highlighting recent progress with Arabidopsis (Arabidopsis thaliana) and raising questions about mitochondrial GABA and succinic semialdehyde (SSA) transport, the fate of succinic semialdehyde once it exits mitochondria, and biochemical interactions between GABA metabolism and related processes such as photorespiration.

Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism.

Proteins of the YgfZ family occur in all domains of life and are characterized by the conserved dodecapeptide motif KGC[Y/F]-x-GQE-x(3) -[R/K]. YgfZ proteins are known to participate in assembly or repair of iron/sulphur clusters, and to require folate for biological activity, but their mechanism of action is unknown. To assess the importance of individual residues in the conserved motif, Escherichia coli Ygf Z was expressed from a plasmid in a ΔygfZ strain and subjected to alanine-scanning mutagenesis. The impacts on YgfZ functionality were evaluated by assays of growth and of the in vivo activity of the iron/sulphur enzyme MiaB, which modifies tRNA. By these criteria, the motif's tyrosine residue (Y229) had a detectable influence but only the cysteine residue (C228) was critical, for only the C228A mutant failed to complement the growth and MiaB activity phenotypes of the ΔygfZ strain. Immunoblots confirmed that the latter result was not simply because of a low level of the C228A mutant protein. Collectively, these data demonstrate a pivotal role for the Ygf Z motif's cysteine residue and a subsidiary one for the adjacent tyrosine, and help formulate a hypothesis about the folate requirement of Ygf Z proteins.

Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit.

Little is known about how plants regulate their folate content, including whether the expression of folate biosynthesis genes is orchestrated during development or modulated by folate levels. Nor is much known about how folate levels impact the expression of other genes. These points were addressed using wild-type tomato fruit and fruit engineered for high folate content. In wild-type fruit, the expression of genes specifying early steps in folate biosynthesis declined during development but that of other genes did not. In engineered fruit overexpressing foreign GTP cyclohydrolase I and aminodeoxychorismate synthase genes, the expression of the respective endogenous genes did not change, but that of three downstream pathway genes-aminodeoxychorismate lyase, dihydroneopterin aldolase, and mitochondrial folylpolyglutamate synthase-respectively increased by up to 7.8-, 2.8-, and 1.7-fold, apparently in response to the build-up of specific folate pathway metabolites. These results indicate that, in fruit, certain folate pathway genes are developmentally regulated and that certain others are subject to feedforward control by pathway intermediates. Microarray analysis showed that only 14 other transcripts (of 11 000 surveyed) increased in abundance by two-fold or more in high-folate fruit, demonstrating that the induction of folate pathway genes is relatively specific.

Metabolism of the folate precursor p-aminobenzoate in plants: glucose ester formation and vacuolar storage.

Plants produce p-aminobenzoate (pABA) in chloroplasts and use it for folate synthesis in mitochondria. In plant tissues, however, pABA is known to occur predominantly as its glucose ester (pABA-Glc), and the role of this metabolite in folate synthesis has not been defined. In this study, the UDP-glucose:pABA acyl-glucosyltransferase (pAGT) activity in Arabidopsis extracts was found to reside principally (95%) in one isoform with an apparent K(m) for pABA of 0.12 mm. Screening of recombinant Arabidopsis UDP-glycosyltransferases identified only three that recognized pABA. One of these (UGT75B1) exhibited a far higher k(cat)/K(m) value than the others and a far lower apparent K(m) for pABA (0.12 mm), suggesting its identity with the principal enzyme in vivo. Supporting this possibility, ablation of UGT75B1 reduced extractable pAGT activity by 95%, in vivo [(14)C]pABA glucosylation by 77%, and the endogenous pABA-Glc/pABA ratio by 9-fold. The K(eq) for the pABA esterification reaction was found to be 3 x 10(-3). Taken with literature data on the cytosolic location of pAGT activity and on cytosolic UDP-glucose/UDP ratios, this K(eq) value allowed estimation that only 4% of cytosolic pABA is esterified. That pABA-Glc predominates in planta therefore implies that it is sequestered away from the cytosol and, consistent with this possibility, vacuoles isolated from [(14)C]pABA-fed pea leaves were estimated to contain> or =88% of the [(14)C]pABA-Glc formed. In total, these data and the fact that isolated mitochondria did not take up [(3)H]pABA-Glc, suggest that the glucose ester represents a storage form of pABA that does not contribute directly to folate synthesis.

Cloning and characterization of two NAD kinases from Arabidopsis. identification of a calmodulin binding isoform.

NAD kinase (NADK; ATP:NAD 2'-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca2+-dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaM-dependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (Km NAD=0.20 mM, Km Mg2+ -ATP=0.17 mM). The enzyme was fully activated by conserved CaM (S0.5 = 2.2 nm) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins. Possible roles for NADKs in plants are discussed in light of our observations.