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Biochem. J. (2003) 370 (505–516) (Printed in Great Britain)

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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* , Helen K. LEECH* , Richard BECK* , Heidi L. SCHUBERT† , Patricio J. SANTANDER‡ , Charles A. ROESSNER‡ , A. Ian SCOTT‡ , Jan H. MARTENS§ , Dieter JAHN§ , Claude THERMES , Alain RAMBACH¶ and Martin J. WARREN*¹

*School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, U.K., †Department of Biochemistry, University of Utah, Salt Lake City, UT 84132, U.S.A., ‡Center for Biological NMR, Department of Chemistry, Texas A & M University, College Station, Texas 77843, U.S.A., §Institute for Microbiology, Technical University Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany, Centre de Génétique Moléculaire, Laboratoire associé à l'Université Pierre et Marie Curie, Centre National de la Recherche Scientifique, 91198 Gif-sur-Yvette, France, and ¶CHROMagar, 4 Place du 18 Juin 1940, 75006 Paris, France

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.

Key words: CbiX, SirA, SirB, SirC, sirohydrochlorin.

Abbreviations used: IPTG, isopropyl b-D-thiogalactoside; LB, Luria–Bertani (broth); PBG, porphobilinogen; SAM, S-adenosyl-L-methionine.

¹To whom correspondence should be addressed (e-mail m.j.warren@qmul.ac.uk).

Sequence data for B. megaterium hemAXBCDL have been deposited with the accession number AJ508220.

Received 16 September 2002/21 October 2002; accepted 31 October 2002

Published as BJ Immediate Publication 31 October 2002, DOI 10.1042/BJ20021443

The Biochemical Society, London © 2003

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