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J. Biol. Chem., Vol. 283, Issue 16, 10813-10821, April 18, 2008

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Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis^*

Andrew D. Lawrence, Evelyne Deery, Kirsty J. McLean, Andrew W. Munro, Richard W. Pickersgill^¶1, Stephen E. J. Rigby^¶, and Martin J. Warren²

From the Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, and ^¶School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom

Vitamin B₁₂, the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6Å resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction.

Received for publication, December 21, 2007 , and in revised form, February 7, 2008.

The atomic coordinates and structure factors (code 3CB0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Biotechnology and Biological Sciences Research Council and Queen Mary, University of London, in the form of a studentship (to A. D. L.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, additional references, and Figs. S1-S6.

¹ To whom correspondence may be addressed: School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Rd., London E1 4NS, UK. Tel.: 44-20-7882-6360; E-mail: r.w.pickersgill{at}qmul.ac.uk.

² To whom correspondence may be addressed: Dept. of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK. Tel.: 44-1227-824690; E-mail: m.j.warren{at}kent.ac.uk.

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