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 con- firmed 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 aconi- tase. When successive steps in folate biosynthesis were ablated, ∆folE (lacking pterins and folates) and ∆folP (lacking folates) mu- tants mimicked the ∆ygfZ mutant in having low MiaB activities, whereas ∆folE ∆thyA mutants supplemented with 5-formyltetrahy- drofolate (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 plum- bagin 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.