A Role for Tetrahydrofolates in the Metabolism of Iron-Sulfur Clusters in All Domains of Life

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

Jeffrey C. Wallera, Sophie Alvarezb, Valeria Naponellic, Aurora Lara-Nuñezc, Ian K. Blabyd, Vanessa Da Silvac, Michael J. Ziemaka, Tim J. Vickerse, Stephen M. Beverleye, Arthur S. Edisonf, James R. Roccag, Jesse F. Gregory IIIc, Valérie de Crécy-Lagardd, and Andrew D. Hansona,1

aHorticultural Sciences, cFood Science and Human Nutrition, dMicrobiology and Cell Science Departments, and fDepartment of Biochemistry and Molecular Biology and National High Magnetic Field Laboratory, and gMcKnight Brain Institute, University of Florida, Gainesville, FL 32611; bDonald Danforth Plant Science Center, Saint Louis, MO 63132; and eDepartment of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110

Edited* by Rowena G. Matthews, University of Michigan, Ann Arbor, MI, and approved March 8, 2010 (received for review October 7, 2009)

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 re- quire 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. In- activating 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 inter- Fig. 1. Structure of tetrahydrofolate (THF) and its C1 derivatives. The mono- mediate MiaB activities. These data indicate that YgfZ requires a glutamyl form is shown; a short γ-linked polyglutamyl tail is usually attached folate, most probably tetrahydrofolate. Importantly, the ∆ygfZ to the γ-carboxyl of the glutamate. C1 groups at various levels of oxidation mutant was hypersensitive to oxidative stress and grew poorly are attached to N-5 and/or N-10 of THF as shown. on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of protein of the radical S-adenosylmethionine (SAM) family whose these growth phenotypes as well as the MiaB activity phenotype. activity drops in mutants defective in Fe/S cluster synthesis (10). Comparative genomic analysis indicated widespread functional The selectivity of these activity losses places Iba57p, and possibly associations between COG0354 proteins and Fe/S cluster metabo- YgfZ, in the category of proteins affecting particular subsets of lism. Thus COG0354 proteins have an ancient, conserved, folate- Fe/S enzymes. Both Iba57p and YgfZ form complexes with IscA- dependent function in the activity of certain Fe/S cluster enzymes. type proteins (7 and 11). Like other proteins involved in Fe/S cluster synthesis or repair, YgfZ is induced by oxidative stress comparative genomics ∣ oxidative stress ∣ YgfZ protein ∣ Iba57 ∣ COG0354 (12). Some bacterial COG0354 genes are apparently essential, although E. coli ygfZ is not (9 and 13). ron-sulfur (Fe/S) clusters are versatile but labile prosthetic COG0354 proteins are structurally similar to folate-dependent Igroups found in >100 proteins from all domains of life (1 and enzymes including glycine decarboxylase T protein (GcvT), 2). Fe/S cluster synthesis, assembly, and repair have consequently dimethylglycine and sarcosine oxidases, and TrmE (14 and 15), drawn much attention, and genetic and biochemical approaches all of which mediate one-carbon (C1) transfer reactions involving have begun to dissect the multicomponent systems involved. It tetrahydrofolate (THF) (Fig. 1). Like the three-dimensional is now known that the required sulfur is generated in persulfide structures of these proteins, that of YgfZ predicts a folate-binding form by cysteine desulfurylases (e.g., IscS, SufSE) and then trans- site (14). ferred to scaffold proteins (e.g., IscU, NfuA) on which clusters In this study, we used structural, biochemical, and mutational are assembled and from which they are mobilized to target apo- approaches to show that YgfZ binds folate in vitro, that folate proteins (1–4). However, many aspects of synthesis and assembly synthesis mutations mimic the effect of ablating YgfZ, and that remain opaque, including the roles of auxiliary proteins that are YgfZ can be functionally replaced by COG354 proteins from all needed for the activity of various subsets of Fe/S enzymes (1–4). domains of life. Repair of Fe/S clusters is less understood but is clearly vital during oxidative stress (5 and 6). Author contributions: J.C.W., V.d.C.-L., and A.D.H. designed research; J.C.W., S.A., V.N., A surprising new player in the Fe/S cluster arena is the protein A.L.-N., I.K.B., V.D.S., M.J.Z., T.J.V., S.M.B., A.S.E., J.R.R., J.F.G., V.d.C.-L., and A.D.H. family classified as COG0354 in the Clusters of Orthologous performed research; A.S.E. and J.R.R. analyzed data; and A.D.H. wrote the paper. Groups database. COG0354 proteins occur in all domains of life The authors declare no conflict of interest. and include yeast Iba57p and Escherichia coli YgfZ. Inactivating *This Direct Submission article had a prearranged editor. iba57, whose product is mitochondrial, resulted in activity loss in Freely available online through the PNAS open access option. four Fe/S enzymes and a petite phenotype (7 and 8). Similarly, 1 To whom correspondence should be addressed. E-mail: [email protected]. ablating ygfZ decreased the MiaB-mediated methylthiolation This article contains supporting information online at www.pnas.org/lookup/suppl/ 6 of N -isopentenyladenosine in tRNA (9), MiaB being an Fe/S doi:10.1073/pnas.0911586107/-/DCSupplemental.

10412–10417 ∣ PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 www.pnas.org/cgi/doi/10.1073/pnas.0911586107 Downloaded by guest on October 1, 2021 able estimates of the equilibrium dissociation constant (KD) for (6S)-5-formyl-THF binding to YgfZ would accordingly be k as low as 0.1 mM for an association rate constant ( on) limited 107 −1 −1 k by diffusion ( M s ) or as high as 3 mM if on were an order of magnitude slower (106 M−1 s−1). Tryptophan fluorescence quenching (14) also indicated that YgfZ binds folates, but the data were not corrected for the inner filter effect and so required corroboration.

Ablating YgfZ in E. coli Impacts Multiple Fe/S Enzymes. Totest whether deleting ygfZ has effects beyond that on MiaB (9) we compared the activities of six diverse Fe/S enzymes in wild type and ΔygfZ strains (Table 1) grown without or with oxidative stress imposed with the redox-cycling agent plumbagin (5-hydroxy-2-methyl- 1,4-naphthoquinone) (12). Succinate dehydrogenase, fumarase, and dimethylsulfoxide reductase activities were all significantly lower in the ΔygfZ strain, especially under oxidative stress, and the same is presumably true of 6-phosphogluconate dehydratase as this strain failed to use gluconate as carbon source when plumbagin was present (Table 1). Aconitase B was unaffected by the deletion, and sulfite reductase activity was mildly increased, showing that the negative effect of deleting ygfZ is specific to Fig. 2. NMR evidence for folate binding by E. coli YgfZ. Samples for each certain enzymes. spectrum contained 1 mM (6S)-5-formyl-THF with 13C labels in the glutamate moiety. One-dimensional 13C-HSQC experiments obtained with 512 scans provided a filter to observe only 1H resonances directly bonded to 13C. Mutational Ablation of Folate Pools. The folate requirement of (A)(6S)-5-Formyl-THF alone; (B)(6S)-5-formyl-THF plus 1 mM YgfZ; (C)(6S)- E. coli YgfZ could in principle be probed by blocking various 5-formyl-THF plus 1 mM YgfZ and 9 mM unlabeled (6R)-5-formyl-THF; steps in folate biosynthesis (Fig. 3A) and measuring the activity (D)(6S)-5-formyl-THF plus 1 mM YgfZ and 9 mM unlabeled (6S)-5-formyl- of a YgfZ-dependent “reporter” Fe/S enzyme. To validate this THF. Corresponding full one-dimensional 1H spectra are shown in Fig. S1, approach we analyzed folates in a set of strains respectively and the 2D HSQC-TOCSY spectrum of 5-formyl-THF is in Fig. S2. The large designed to eliminate: (i) folates and their pterin precursors ∼3 4 cluster of resonances from . to 3.6 ppm is attributable to glycerol (ΔfolE), (ii) folates but not pterins (ΔfolP), (iii) essentially all and triethylene glycol in the buffer. No interaction with YgfZ was seen when Δ Δ 13C-labeled folic acid (1 mM) replaced labeled (6S)-5-formyl-THF. C1-substituted folates ( gcvP glyA), or (iv) dihydrofolate (DHF) and pterins (a ΔfolE ΔthyA strain expressing Synechocystis Results folate carrier Slr0642 and given 5-formyl-THF; 16). Results were as predicted although the latter strain had 5-fold less total folate

YgfZ Shows Folate-Binding in Vitro. NMR was used to monitor BIOCHEMISTRY than the wild type (Table 2). The ΔgcvP ΔglyA strain lacked the interaction of purified YgfZ with the natural (6S) form of detectable C1 folates, and was functionally C1-folate deficient 5-formyltetrahydrofolate (5-formyl-THF) or folic acid, both 13 as it did not support synthesis of the tRNA base 5-methylamino- labeled in the glutamate moiety with C. Clear interaction 5 2 methyl-2-thiouridine (mnm s U), which requires a C1 folate (17) was observed with (6S)-5-formyl-THF; this interaction was abol- (Fig. 3B). Nor was mnm5 s2 U detected in other folate-deficient ished by adding a 9-fold excess of unlabeled (6S)-5-formyl-THF strains (Fig. 3B). Pterin analysis confirmed the presence of pterins but not (6R)-5-formyl-THF (Fig. 2, Fig. S1, Fig. S2). The small in the ΔfolP strain, the most prominent being monapterin (18). effect of the 6R form may be due partly to contaminating 6S. These data indicate stereoselective binding of 5-formyl-THF; YgfZ Activity is Folate-Dependent. Having confirmed the predicted no interaction was observed with folic acid. The complete loss effects on folate pools, we used MiaB as a reporter to assess of resonances in Fig. 2B (5-formyl-THF plusYgfZ) results from whether YgfZ activity requires a folate and, if so, which one(s). intermediate exchange. Assuming the difference between free MiaB mediates posttranscriptional methylthiolation of N6- and YgfZ-bound chemical shifts of 5-formyl-THF to be 1 ppm, isopentenyladenosine (i6A) in tRNAs, giving 2-methylthio- the data would fit with an exchange rate of ∼3; 700 Hz. Reason- N6-isopentenyladenosine (ms2i6A), so that reducing MiaB

Table 1. Effect of deleting ygfZ on Fe/S enzyme activities in E. coli Activity, nmol min−1 mg−1 protein Control +Plumbagin 25 μM Enzyme Wild type ΔygfZ Wild type ΔygfZ Succinate dehydrogenase 63.0 ± 3.4 33.1 ± 11.1* (−47%)* 57.4 ± 6.9 16.8 ± 2.6** (−71%) Fumarase A + B 1,092 ± 110 742 ± 86** (−32%) 1,431 ± 176 862 ± 179*** (−40%) DMSO reductase† 48.0 ± 7.9 0.8 ± 0.1 (−98%) 16.5 ± 2.4 <0.005*(−100%) 6-P-Gluconate 319 ± 74 363 ± 98 195 ± 60 ND‡ dehydratase Aconitase B 46.1 ± 4.2 49.4 ± 4.3 53.6 ± 1.1 45.4 ± 9.5 Sulfite reductase 680 ± 30 805 ± 36* (+18%) 1,281 ± 91 1,673 ± 122* (+31%) Data are means of 2–6 independent replicates SE. *, **, and *** denote differences between wild type and deletant that are significant at P < 0.05, <0.01, and <0.001, respectively. *Values in parentheses are percent change in activity in the deletant relative to the wild type. †Measured in vivo in aerobically grown cultures; values are consequently not directly comparable to others in the table. ‡Not determined; the ΔygfZ strain was unable to grow on the gluconate-containing induction medium in the presence of 25 μM plumbagin.

Waller et al. PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 ∣ 10413 Downloaded by guest on October 1, 2021 activity in the ΔfolE ΔthyA strain may have been below wild type due to low folate levels (Table 2). The ΔfolE ΔthyA strain given 5-formyl-THF cannot synthesize DHF enzymatically (Fig. 3A) and none was detected (Table 2). The high MiaB activity in this strain (Fig. 3C) therefore argues against a DHF requirement, and so narrows the field of possibi- lities to THF or one of its C1 forms. That the requirement can be met by THF itself is suggested by the intermediate MiaB activity of the ΔgcvP ΔglyA strain, which contains THF but no C1 folates (Table 2 and Fig. 3B). MiaB activity—and hence YgfZ activity—in this strain may not have reached that of the wild type because its 18-fold elevated THF level (Table 2) caused substrate inhibition (19).

ygfZ Deletion Impairs Oxidative Stress Resistance and Growth on Mini- mal Media. As YgfZ is induced by oxidative stress (12) we tested the sensitivity of the ΔygfZ strain to plumbagin or paraquat added to LB medium. The deletant showed only a slight growth defect on LB alone but was more sensitive to both stress agents (Fig. S3A). It also grew poorly on minimal A salts or M9 medium with glucose, glycerol, or acetate as carbon source (Fig. S3B); this fits with the lowered activities of the Krebs cycle enzymes succinate dehydrogenase and fumarase (Table 1).

Eukaryotic and Prokaryotic COG0354 Genes Complement the ygfZ Mutation. The above growth phenotypes allowed complementation tests of COG0354 genes from bacterial, archaeal, protist, fungal, animal, and plant sources. Two plant genes were tested, Arabidopsis At4g12130 and At1g60990, whose products are respectively predicted to be mitochondrial and plastidial. All seven genes tested complemented the oxidative stress hypersensitivity phenotype (Fig. 4A). The bacterial, Leishmania, mouse, and Arabidopsis genes also complemented the growth phenotype Fig. 3. Mutational analysis of the folate requirement of E. coli YgfZ. on minimal medium, but the archaeal gene did not and the yeast (A) Outline of THF biosynthesis and C1-folate metabolism showing the muta- gene did so only weakly. As the complementation results were tional strategy and its predicted effects on folate pools. DHF, dihydrofolate; obtained in darkness, COG0354 action cannot depend upon light. p H2 pterins, dihydropterins; H2 pteroate, dihydropteroate; pABA, -amino- To check that eukaryotic COG0354 genes also complemented benzoate. (B) LC-MS/MS quantification of the tRNA nucleoside 5-methylami- 2 6 ∕ 6 nomethyl-2-thiouridine (mnm5s2U) in wild type and deletant strains grown the MiaB activity phenotype, the ms i A i A ratio was deter- in Antibiotic Medium 3 supplemented as described in Materials and Methods. mined for cells harboring the genes from mouse or Leishmania Data are means and standard errors for three independent samples. (Fig. 4B, left). Both genes greatly increased the ratio compared to (C) LC-MS/MS quantification of the tRNA nucleosides i6A and ms2i6A in wild the vector-only control. To probe folate dependency, the mouse type and deletant strains, and the ms2i6A∕i6A ratio. Other details as in B. and Leishmania genes were tested in a ΔfolP ΔygfZ background. The ms2i6A∕i6A ratio for both remained very low, thereby impli- cating folates in their activity (Fig. 4B, right). Parenthetically, this 2 6 ∕ 6 activity decreases the ms i A i A ratio (9 and 10). This ratio was assay does not test the activity of the mouse or Leishmania pro- measured in the four strains described above and in ygfZ and ’ 2 6 teins with these species native pterin cofactor tetrahydrobiopter- miaB deletants as benchmarks; the latter forms no ms i A in since E. coli has tetrahydromonapterin as its predominant (Fig. 3C). Deleting folE lowered MiaB activity almost as much pterin (18 and 20). as deleting ygfZ itself, which establishes pterin or folate dependency. A role for pterins was excluded because: (i) the folP Comparative Genomics Links COG0354 to Fe/S Clusters, Folates, and deletant—which has pterins but no folates—showed greatly Oxygen. Analysis of the phyletic distribution and chromo- lowered MiaB activity, and (ii) the ΔfolE ΔthyA strain given somal clustering of prokaryotic COG0354 genes showed that they 5-formyl-THF—which has folates but no pterins—did not. MiaB cooccur (i.e., are present or absent together) with genes encoding

Table 2. Folate profiles of E. coli strains Folates, pmol mg−1 protein*

† Strain THF CH3-THF CH¼THF þ 10-CHO-DHF 5-CHO-THF Total Wild type 48.1 ± 10.7 10.6 ± 1.9 738 ± 93 68.9 ± 10.9 866 ± 114 ΔfolE <0.05 <0.05 <0.05 <0.05 <0.2 ΔfolP <0.05 <0.05 <0.05 <0.05 <0.2 ΔgcvP ΔglyA 845 ± 171 <0.05 <0.05 <0.05 845 ± 171 ΔfolEΔthyA þ 5-CHO-THF‡ 152 ± 100 7.1 ± 0.7 14.4 ± 3.5 5.8 + 1.5 180 ± 98

*Means and standard errors of 3–7 replicates. THF, tetrahydrofolate; CH3-THF, 5-methyl-THF; CH¼THF, 5,10-methenyl-THF; 10-CHO-DHF, 10-formyl-DHF; 5-CHO-THF, 5-formyl-THF. The detection limit for each folate was 0.05 pmol mg−1 protein. †10-CHO-THF is converted to CH ¼ THF and 10-CHO-DHF during analysis. ‡Dihydrofolate not detected (detection limit 0.25 pmol mg−1 protein, allowing for 20% recovery).

10414 ∣ www.pnas.org/cgi/doi/10.1073/pnas.0911586107 Waller et al. Downloaded by guest on October 1, 2021 enzyme activity data extend the known protein repertoire of COG0354 proteins (7 and 9), and the chromosomal clustering data suggest this repertoire may be wider still. The complementation results extend the species repertoire by showing that eukaryotic, archaeal, and bacterial COG0354 genes can replace E. coli ygfZ in some conditions. A human gene was similarly shown to replace iba57 in yeast (7). The NMR evidence for folate binding came from (6S)-5- formyl-THF, THF itself being too unstable for such studies. While not a C1 donor, (6S)-5-formyl-THF binds to (and inhibits) most folate-dependent enzymes and is often used as a stable surro- gate for THF (15). The estimated KD (≥0.1 mM) for (6S)-5- formyl-THF is higher than typically seen for binding of folate substrates. This higher KD could arise because 5-formyl-THF is not the natural YgfZ substrate, or due to the absence of polyglu- tamylation, which typically lowers KD values. Moreover, since YgfZ normally occurs in complexes with IscA-type proteins in vivo (7 and 11), the free protein could adopt a nonnative conformation, as seen with the paralogous folate-binding protein GcvT (23). A nonnative conformation would be likely to affect ligand binding kinetics. Thus we conclude that COG0354 has a folate-dependent function that impacts diverse Fe/S proteins and is at least partly conserved in all domains of life. Our results shed light on the nature of this folate-dependent function: First, the genomic co- distribution data imply that COG0354 proteins cooperate with IscA-SufA family proteins, in agreement with the finding that yeast and E. coli COG0354 proteins complex with IscA homologs (7 and 11). Second, the experimental and genomic data connect- ing COG0354 proteins with oxidative stress, and the nonuniform effects on various Fe/S enzyme activities, point to a role in repair of specific enzymes as opposed to de novo synthesis of all Fe/S clusters. Third, while both YgfZ and Iba57p clearly affect only Fig. 4. Complementation of the ygfZ mutation by diverse COG0354 subsets of the Fe/S enzymes in their respective host organism, Δ genes. (A) Tests with the ygfZ strain harboring pBAD24 alone (Vector) or these subsets appear not to be identical; most notably, aconitase expressing E. coli YgfZ or COG0354 proteins from a bacterium phylogeneti- cally distant from E. coli (Bartonella henselae), from the eukaryotes was impacted by COG0354 ablation in yeast (7) but not E. coli. BIOCHEMISTRY Arabidopsis (m, predicted mitochondrial; c, predicted plastidial), mouse, Another hint of variation in target enzyme specificity is that all Leishmania major, and Saccharomyces cerevisiae, and from the archaeon the COG0354 genes tested complemented the ΔygfZ mutant Haloferax volcanii. Three independent clones were streaked of each under oxidative stress but not all did so on minimal medium. construct. Plates contained LB medium minus or plus 30 μM plumbagin, or Different Fe/S proteins are presumably crucial to growth in these M9 medium with 0.2% glycerol, 0.02% arabinose, and appropriate antibio- contrasting conditions. Supposing a repair role, the enzymes tics. Incubation was at 37 °C for 2 d (LB) or at 22 °C for 5 d (M9). (B) LC-MS/MS 6 2 6 2 6 6 targeted in any given organism could reflect idiosyncrasies in quantification of i A and ms i A, and the ms i A∕i A ratio, in tRNA of the the susceptibility of different Fe/S clusters to damage, as seen Δ Δ Δ ygfZ or folP ygfZ strains harboring vector alone or expressing mouse or in other studies (5, 6, 10). Leishmania major COG0354, grown in Antibiotic Medium 3 plus 300 μM thymidine, 0.02% arabinose and appropriate antibiotics. Data are means What role might THF play in the function of COG0354? One and standard errors for three independent samples. possibility that we consider remote is a purely structural role, as with folate hexaglutamate in the assembly of bacteriophage T4 (24). A second and more likely possibility is that THF acts as Fe/S assembly proteins of the IscA-SufA family in 90% of the an electron donor, there being reductions in Fe/S cluster assembly genomes examined, and never occur without them (Fig. S4A). and repair whose electron donor is unknown (3 and 5). THF can IscA-SufA genes themselves occur in only 69% of genomes while be oxidized readily (standard redox potential ¼ −230 mV for other genes of the Isc and Suf systems are nearly universal the DHF/THF pair) and the redox properties of the THF (Fig. S3A). COG0354 genes also often cluster with genes for ring are exploited in nature in thymidylate synthesis, where 5, Fe/S enzymes and related proteins (Table S1 and Fig. S4B). Con- 10-methylenetetrahydrofolate acts as both electron donor and necting COG0354 to folates, COG0354 genes in Archaea are C1 donor (25). There is also precedent for THF acting solely confined to Halobacteria (Fig. S4A), which are almost the only as an electron donor in the case of bacterial phenylalanine hydro- Archaea with folates (21 and 22). Finally, making a link with xylase (26). A third possibility would involve C1 metabolism, oxidative stress, COG0354 genes occur in 86% of aerobes having perhaps with THF acting as an acceptor for a C1 moiety. A C1 a full complement of Fe/S proteins but in only 15% of anaerobes. adduct on an Fe/S cluster-liganding cysteine residue would block Furthermore, the E. coli ygfZ gene contains a marbox structure cluster formation, and might be removed via COG0354-mediated and is SoxS regulated (12). transfer to THF in a mechanism analogous to that of the glycine Discussion cleavage T protein or oxidative demethylases. Future studies will Our data establish a unique role for folate as a cofactor for E. coli address these alternative propositions. YgfZ in the assembly or repair of Fe/S clusters, and confirm a Materials and Methods similar role in mouse and Leishmania COG0354 proteins. The Bioinformatics. Prokaryote genomes were analyzed using the SEED database data also demonstrate the criticality of YgfZ during oxidative and its tools (27). Full results are available at http://theseed.uchicago.edu/FIG/ stress, in agreement with its induction in such conditions (12) in the YgfZ and Iron-sulfur cluster assembly subsystems. Eukaryote genomes and with the rarity of COG0354 genes in anaerobes. The Fe/S and ESTs were searched using BLAST algorithms at NCBI (http://www.ncbi.

Waller et al. PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 ∣ 10415 Downloaded by guest on October 1, 2021 nlm.nih.gov/). COG0354 members were identified using the NCBI Conserved spectrometry. Protein was measured by the bicinchoninic acid method with Domain algorithm and the KGCYxGQE motif (14). bovine serum albumin as standard.

E. coli Strains, Plasmids, and Media. Strains, plasmids, and primers are listed Folate and Pterin Analysis. Folates were extracted from cultures grown to an in Table S2 and Table S3. Deletions from the Keio collection (28) and a OD600 of 1.0, deglutamylated, isolated by affinity chromatography, and ΔfolP∷Kan allele (29) were transferred to E. coli K12 MG1655 by P1 transduc- analyzed by HPLC with electrochemical detection as described (20) except tion; for double deletants, Kan cassettes were removed by flippase-mediated that, for DHF analysis, initial extraction was at pH 2.0. Total pterins from recombination. All deletions were PCR-verified. Strains for complementation cultures grown to an OD600 of 1.0 were analyzed as described (20). studies harbored pACYC-RP. Cells were cultured at 37 °C in Antibiotic Medium 3 (Difco) plus 300 μM thymidine, LB medium, minimal A salts (30) or M9 Nucleoside Analysis. Bulk nucleic acids were isolated from stationary phase medium (16) as indicated. The ΔfolE ΔthyA strain harboring plasmid-borne cells and enriched for tRNA (38) before Nucleobond AXR 400 column slr0642 was given 50 μM 5-formyl-THF, 1 mM DTT, and 0.1% (w/v) ascorbate. chromatography purification (Machery-Nagel). Pure tRNA was then hydro- Antibiotic concentrations (μg/mL) were: kanamycin, 25; chloramphenicol, 10; lyzed and analyzed by liquid chromatography-tandem mass spectrometry ampicillin, 25; tetracycline, 10; erythromycin, 200. Gene expression was (LC-MS/MS) as described (39). These procedures differ from those of Ote induced with 0.02% (w/v) arabinose or 1 mM isopropyl-β-D-thiogalactoside. et al. (9), which may account for differences in our ms2i6A∕i6A ratios. COG0354 sequences are described in Table S4. 13 NMR Analyses. (6S)-5-Formyl-THF [ C5-glutamate] was from Merck Eprova; Fe/S Enzyme Assays. Wild type and ΔygfZ strains were grown aerobically, other folates were from Schircks. 1-mm NMR tubes were used in a 5-mm cryo- minus or plus 25 μM plumbagin, to an OD600 of ∼1 in LB medium except genic probe on a Bruker AvanceII 600 MHz instrument. Sample volumes were for 6-phosphogluconate dehydratase, which was induced in minimal A salts 10 μL for each experiment; temperature was 20 °C. One-dimensional 13C-het- containing 0.2% (w/v) K-gluconate. Enzymes were assayed at 22 °C. Succinate eronuclear single quantum coherence (HSQC) experiments were used to dehydrogenase was assayed in inverted membrane vesicles (31) after activat- select for protons directly bonded to 13C. Due to the low volume, 512 ing in 0.6 M K-phosphate, pH 7.0, at 30 °C for 20 min. Assays contained 0.1 M scans were collected for each experiment. One-dimensional 1H spectra were K-phosphate, pH 7.8, 10 mM succinate, 0.75 mM phenazine methosulfate, collected with 16 scans per sample. For more details, see SI Text. 30 μM 2,6-dichlorophenolindophenol, and 1 mM KCN; dye reduction was ε ¼ 21 −1 −1 monitored at 600 nm ( mM cm ). Soluble extracts were prepared ACKNOWLEDGMENTS. We thank G. Swedberg for the ΔfolP∷Kan strain; as described (32). Aconitase was assayed spectrophotometrically in mixtures D. Tieman and M. Bailly for assistance; K. Austin and D. Fremont for efforts þ containing 50 mM Tris-HCl, pH 7.6, 25 mM Na citrate, 0.25 mM NADP , to test folate binding by surface plasmon resonance; J. Peng for advice on 0.6 mM MnCl2, 1-2 units of isocitrate dehydrogenase in a total volume of binding constant estimates; and S. Jang for criticism of the manuscript. This 0.1 mL (33). The activity sensitive to 1 mM EDTA was taken as that of aconi- work was supported in part by the National Science Foundation award tase B (34). Fumarase A plus B activity was assayed as described (35) in 50 mM MCB-0839926 (to A.D.H.), by the Department of Energy award FG02- Tris-HCl, pH 7.6. 6-Phosphogluconate dehydratase was assayed as described 07ER64498 (to V.d.C.-L.), by National Institutes of Health Grant AI21903 (to S.M.B.), by EMBO fellowship ALTF 106-2005 (to T.J.V.), and by an endow- (36). Sulfite reductase was assayed spectrophotometrically in 50 mM Tris-HCl, ment from the C.V. Griffin, Sr. Foundation. NMR studies were supported pH 7.5, containing 0.25 mM NADPH and 0.3 mM ferricyanide (37). Dimethyl- by the National Science Foundation’s National High Magnetic Field Labora- sulfoxide reductase was assayed in vivo by growing cultures supplied with tory User Program in the Advanced Magnetic Resonance Imaging and 14 mM DMSO to stationary phase, and determining the rate of dimethylsul- Spectroscopy (AMRIS) Facility in the McKnight Brain Institute of the Univer- fide production by headspace analysis using gas-chromatography/mass sity of Florida.

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