enzyme recognition sites on the primers listed are in lower case letters. Quikchange mutagenesis was performed using the corresponding primers to generate the variant construct from the parental wild-type construct. Mutation sites that are not complementary to the parental construct on the primers are in lower case letters. (DOC) References 1. Waugh R, Boxer DH (1986) Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity. Biochimie 68: 157–166. 2. Jacobi A, Rossmann R, Bo¨ck A (1992) The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli. Arch Microbiol 158: 444–451. 3. Maier T, Lottspeich F, Bo¨ck A (1995) GTP hydrolysis by HypB is essential for nickel insertion into hydrogenases of Escherichia coli. Eur J Biochem 230: 133–138. 4. Olson JW, Fu CL, Maier RJ (1997) The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase. Mol Microbiol 24: 119–128. 5. Olson JW, Mehta NS, Maier RJ (2001) Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori. Mol Microbiol 39: 176–182. 6. Hube M, Blokesch M, Bock A (2002) Network of hydrogenase maturation in Escherichia coli: role of accessory proteins HypA and HybF. J Bacteriol 184: 3879–3885. 7. Hoffmann D, Gutekunst K, Klissenbauer M, Schulz-Friedrich R, Appel J (2006) Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803. Additional homologues of hypA and hypB are not active in hydrogenase maturation. FEBS J 273: 4516–4527. 8. Bo¨ ck A, King PW, Blokesch M, Posewitz MC (2006) Maturation of hydrogenases. Adv Microb Physiol 51: 1–71. 9. Blokesch M, Bo¨ck A (2002) Maturation of [NiFe]-hydrogenases in Escherichia coli: the HypC cycle. J Mol Biol 324: 287–296. 10. Lo¨scher S, Zebger I, Andersen LK, Hildebrandt P, Meyer-Klaucke W, et al. (2005) The structure of the Ni-Fe site in the isolated HoxC subunit of the hydrogen-sensing hydrogenase from Ralstonia eutropha. FEBS Lett 579: 4287–4291. 11. Winter G, Buhrke T, Lenz O, Jones AK, Forgber M, et al. (2005) A model system for [NiFe] hydrogenase maturation studies: Purification of an active site- containing hydrogenase large subunit without small subunit. FEBS Lett 579: 4292–4296. 12. Paschos A, Glass RS, Bo¨ck A (2001) Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases. FEBS Lett 488: 9–12. 13. Paschos A, Bauer A, Zimmermann A, Zehelein E, Bo¨ck A (2002) HypF, a carbamoyl phosphate-converting enzyme involved in [NiFe] hydrogenase maturation. J Biol Chem 277: 49945–49951. 14. Reissmann S, Hochleitner E, Wang H, Paschos A, Lottspeich F, et al. (2003) Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands. Science 299: 1067–1070. 15. Blokesch M, Albracht SP, Matzanke BF, Drapal NM, Jacobi A, et al. (2004) The complex between hydrogenase-maturation proteins HypC and HypD is an intermediate in the supply of cyanide to the active site iron of [NiFe]- hydrogenases. J Mol Biol 344: 155–167. 16. Roseboom W, Blokesch M, Bo¨ck A, Albracht SP (2005) The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different. FEBS Lett 579: 469–472. 17. Forzi L, Hellwig P, Thauer RK, Sawers RG (2007) The CO and CN(-) ligands to the active site Fe in [NiFe]-hydrogenase of Escherichia coli have different metabolic origins. FEBS Lett 581: 3317–3321. HypA/HypB Interaction and Hydrogenase Maturation Author Contributions Conceived and designed the experiments: KHC KBW. Performed the experiments: KHC KML. Analyzed the data: KHC KML KBW. Contributed reagents/materials/analysis tools: KHC KML. Wrote the paper: KHC KBW. 18. Lenz O, Zebger I, Hamann J, Hildebrandt P, Friedrich B (2007) Carbamoyl- phosphate serves as the source of CN(-), but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. FEBS Lett 581: 3322–3326. 19. Theodoratou E, Huber R, Bo¨ck A (2005) [NiFe]-Hydrogenase maturation endopeptidase: structure and function. Biochem Soc Trans 33: 108–111. 20. Mehta N, Olson JW, Maier RJ (2003) Characterization of Helicobacter pylori nickel metabolism accessory proteins needed for maturation of both urease and hydrogenase. J Bacteriol 185: 726–734. 21. Atanassova A, Zamble DB (2005) Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. J Bacteriol 187: 4689–4697. 22. Watanabe S, Arai T, Matsumi R, Atomi H, Imanaka T, et al. (2009) Crystal structure of HypA, a nickel-binding metallochaperone for [NiFe] hydrogenase maturation. J Mol Biol 394: 448–459. 23. Xia W, Li H, Sze KH, Sun H (2009) Structure of a nickel chaperone, HypA, from Helicobacter pylori reveals two distinct metal binding sites. J Am Chem Soc 131: 10031–10040. 24. Leach MR, Sandal S, Sun H, Zamble DB (2005) Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB. Biochemistry 44: 12229–12238. 25. Dias AV, Mulvihill CM, Leach MR, Pickering IJ, George GN, et al. (2008) Structural and biological analysis of the metal sites of Escherichia coli hydrogenase accessory protein HypB. Biochemistry 47: 11981–11991. 26. Olson JW, Maier RJ (2000) Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization. J Bacteriol 182: 1702–1705. 27. Gasper R, Scrima A, Wittinghofer A (2006) Structural insights into HypB, a GTP-binding protein that regulates metal binding. J Biol Chem 281: 27492–27502. 28. Xia W, Li H, Yang X, Wong KB, Sun H (2011) Metallo-GTPase HypB from Helicobacter pylori and Its Interaction with Nickel Chaperone Protein HypA. J Biol ChemEpub. 29. Maier T, Jacobi A, Sauter M, Bo¨ck A (1993) The Product of the HypB Gene, Which Is Required for Nickel Incorporation into Hydrogenases, Is a Novel Guanine Nucleotide-Binding Protein. J Bacteriol 175: 630–635. 30. Cai F, Ngu TT, Kaluarachchi H, Zamble DB (2011) Relationship between the GTPase, metal-binding, and dimerization activities of E. coli HypB. J Biol Inorg Chem 16: 857–868. 31. Chan K-H, Li T, Wong C-O, Wong K-B (2011) Structural Basis for GTP- Dependent Dimerization of Hydrogenase Maturation Factor HypB. PLoS ONE In press. 32. Ballantine SP, Boxer DH (1985) Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12. J Bacteriol 163: 454–459. 33. Zhang JW, Butland G, Greenblatt JF, Emili A, Zamble DB (2005) A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway. J Biol Chem 280: 4360–4366. 34. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006 0008. 35. Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18: 6097–6100. 36. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14: 1188–1190. PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32592