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J. Biol. Chem., Vol. 280, Issue 2, 1086-1094, January 14, 2005

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Identification and Characterization of a Novel Vitamin B₁₂ (Cobalamin) Biosynthetic Enzyme (CobZ) from Rhodobacter capsulatus, Containing Flavin, Heme, and Fe-S Cofactors^*

Helen M. McGoldrick, Charles A. Roessner¶, Evelyne Raux, Andrew D. Lawrence, Kirsty J. McLean||**, Andrew W. Munro||, Stefano Santabarbara, Stephen E. J. Rigby, Peter Heathcote, A. Ian Scott¶, and Martin J. Warren

From the School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom, the ^¶Center for Biological NMR, Department of Chemistry, Texas A & M University, College Station, Texas 77843, and the ^||Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom

One of the most intriguing steps during cobalamin (vitamin B₁₂) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B₁₂ pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B₁₂ itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.

Received for publication, October 19, 2004 , and in revised form, November 1, 2004.

^* This work was supported in part by grants from the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, the European Union (to M. J. W.), and a National Institutes of Health Merit Award (to A. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^** Supported by the European Community through the X-TB program.

Supported by a Royal Society Leverhulme Trust Senior Research Fellowship.

To whom correspondence should be addressed. Tel.: 020-7882-7718; Fax: 020-7882-7609; E-mail: m.j.warren{at}qmul.ac.uk.

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