Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Mar 1;370(Pt 2):505–516. doi: 10.1042/BJ20021443

Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

Evelyne Raux 1, Helen K Leech 1, Richard Beck 1, Heidi L Schubert 1, Patricio J Santander 1, Charles A Roessner 1, A Ian Scott 1, Jan H Martens 1, Dieter Jahn 1, Claude Thermes 1, Alain Rambach 1, Martin J Warren 1
PMCID: PMC1223173  PMID: 12408752

Abstract

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.

Full Text

The Full Text of this article is available as a PDF (415.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blanche F., Debussche L., Thibaut D., Crouzet J., Cameron B. Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans. J Bacteriol. 1989 Aug;171(8):4222–4231. doi: 10.1128/jb.171.8.4222-4231.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chappell James D., Prota Andrea E., Dermody Terence S., Stehle Thilo. Crystal structure of reovirus attachment protein sigma1 reveals evolutionary relationship to adenovirus fiber. EMBO J. 2002 Jan 15;21(1-2):1–11. doi: 10.1093/emboj/21.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Crouzet J., Cauchois L., Blanche F., Debussche L., Thibaut D., Rouyez M. C., Rigault S., Mayaux J. F., Cameron B. Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase. J Bacteriol. 1990 Oct;172(10):5968–5979. doi: 10.1128/jb.172.10.5968-5979.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fu C., Olson J. W., Maier R. J. HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2333–2337. doi: 10.1073/pnas.92.6.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Griffiths L., Cole J. A. Lack of redox control of the anaerobically-induced nirB+ gene of Escherichia coli K-12. Arch Microbiol. 1987 May;147(4):364–369. doi: 10.1007/BF00406134. [DOI] [PubMed] [Google Scholar]
  6. Hansson M., Rutberg L., Schröder I., Hederstedt L. The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J Bacteriol. 1991 Apr;173(8):2590–2599. doi: 10.1128/jb.173.8.2590-2599.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johansson P., Hederstedt L. Organization of genes for tetrapyrrole biosynthesis in gram--positive bacteria. Microbiology. 1999 Mar;145(Pt 3):529–538. doi: 10.1099/13500872-145-3-529. [DOI] [PubMed] [Google Scholar]
  8. Jordan P. M., Berry A. Mechanism of action of porphobilinogen deaminase. The participation of stable enzyme substrate covalent intermediates between porphobilinogen and the porphobilinogen deaminase from Rhodopseudomonas spheroides. Biochem J. 1981 Apr 1;195(1):177–181. doi: 10.1042/bj1950177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kerby R. L., Ludden P. W., Roberts G. P. In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ. J Bacteriol. 1997 Apr;179(7):2259–2266. doi: 10.1128/jb.179.7.2259-2266.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Olson J. W., Fu C., Maier R. J. The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase. Mol Microbiol. 1997 Apr;24(1):119–128. doi: 10.1046/j.1365-2958.1997.3251690.x. [DOI] [PubMed] [Google Scholar]
  11. Outten C. E., O'Halloran T. V. Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science. 2001 Jun 7;292(5526):2488–2492. doi: 10.1126/science.1060331. [DOI] [PubMed] [Google Scholar]
  12. Raux E., Lanois A., Levillayer F., Warren M. J., Brody E., Rambach A., Thermes C. Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli. J Bacteriol. 1996 Feb;178(3):753–767. doi: 10.1128/jb.178.3.753-767.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Raux E., Lanois A., Rambach A., Warren M. J., Thermes C. Cobalamin (vitamin B12) biosynthesis: functional characterization of the Bacillus megaterium cbi genes required to convert uroporphyrinogen III into cobyrinic acid a,c-diamide. Biochem J. 1998 Oct 1;335(Pt 1):167–173. doi: 10.1042/bj3350167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Raux E., Lanois A., Warren M. J., Rambach A., Thermes C. Cobalamin (vitamin B12) biosynthesis: identification and characterization of a Bacillus megaterium cobI operon. Biochem J. 1998 Oct 1;335(Pt 1):159–166. doi: 10.1042/bj3350159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Raux E., McVeigh T., Peters S. E., Leustek T., Warren M. J. The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis. Biochem J. 1999 Mar 15;338(Pt 3):701–708. [PMC free article] [PubMed] [Google Scholar]
  16. Raux E., Schubert H. L., Warren M. J. Biosynthesis of cobalamin (vitamin B12): a bacterial conundrum. Cell Mol Life Sci. 2000 Dec;57(13-14):1880–1893. doi: 10.1007/PL00000670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Raux E., Thermes C., Heathcote P., Rambach A., Warren M. J. A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis. J Bacteriol. 1997 May;179(10):3202–3212. doi: 10.1128/jb.179.10.3202-3212.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rey L., Imperial J., Palacios J. M., Ruiz-Argüeso T. Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis. J Bacteriol. 1994 Oct;176(19):6066–6073. doi: 10.1128/jb.176.19.6066-6073.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robin C., Blanche F., Cauchois L., Cameron B., Couder M., Crouzet J. Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium. J Bacteriol. 1991 Aug;173(15):4893–4896. doi: 10.1128/jb.173.15.4893-4896.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Santander P. J., Roessner C. A., Stolowich N. J., Holderman M. T., Scott A. I. How corrinoids are synthesized without oxygen: nature's first pathway to vitamin B12. Chem Biol. 1997 Sep;4(9):659–666. doi: 10.1016/s1074-5521(97)90221-0. [DOI] [PubMed] [Google Scholar]
  21. Schubert H. L., Raux E., Wilson K. S., Warren M. J. Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis. Biochemistry. 1999 Aug 17;38(33):10660–10669. doi: 10.1021/bi9906773. [DOI] [PubMed] [Google Scholar]
  22. Schubert H. L., Wilson K. S., Raux E., Woodcock S. C., Warren M. J. The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase. Nat Struct Biol. 1998 Jul;5(7):585–592. doi: 10.1038/846. [DOI] [PubMed] [Google Scholar]
  23. Scott A. I., Irwin A. J., Siegel L. M., Schoolery J. N. Sirohydrochlorin. Prosthetic group of a sulfite reductase enzyme and its role in the biosynthesis of vitamin B12. J Am Chem Soc. 1978 Jan 4;100(1):316–318. doi: 10.1021/ja00469a071. [DOI] [PubMed] [Google Scholar]
  24. Scott A. I., Stolowich N. J., Wang J., Gawatz O., Fridrich E., Müller G. Biosynthesis of vitamin B12: factor IV, a new intermediate in the anaerobic pathway. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14316–14319. doi: 10.1073/pnas.93.25.14316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Spencer J. B., Stolowich N. J., Roessner C. A., Scott A. I. The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase. FEBS Lett. 1993 Nov 29;335(1):57–60. doi: 10.1016/0014-5793(93)80438-z. [DOI] [PubMed] [Google Scholar]
  26. Spencer P., Stolowich N. J., Sumner L. W., Scott A. I. Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium. Biochemistry. 1998 Oct 20;37(42):14917–14927. doi: 10.1021/bi981366f. [DOI] [PubMed] [Google Scholar]
  27. Vary P. S. Prime time for Bacillus megaterium. Microbiology. 1994 May;140(Pt 5):1001–1013. doi: 10.1099/13500872-140-5-1001. [DOI] [PubMed] [Google Scholar]
  28. Walker C. J., Willows R. D. Mechanism and regulation of Mg-chelatase. Biochem J. 1997 Oct 15;327(Pt 2):321–333. doi: 10.1042/bj3270321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Warren M. J., Bolt E. L., Roessner C. A., Scott A. I., Spencer J. B., Woodcock S. C. Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein. Biochem J. 1994 Sep 15;302(Pt 3):837–844. doi: 10.1042/bj3020837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Warren M. J., Jordan P. M. Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase. Biochemistry. 1988 Dec 13;27(25):9020–9030. doi: 10.1021/bi00425a021. [DOI] [PubMed] [Google Scholar]
  31. Warren M. J., Stolowich N. J., Santander P. J., Roessner C. A., Sowa B. A., Scott A. I. Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen III methylase. FEBS Lett. 1990 Feb 12;261(1):76–80. doi: 10.1016/0014-5793(90)80640-5. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES