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J. Biol. Chem., Vol. 282, Issue 33, 23957-23969, August 17, 2007

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Elucidation of Substrate Specificity in the Cobalamin (Vitamin B₁₂) Biosynthetic Methyltransferases

STRUCTURE AND FUNCTION OF THE C20 METHYLTRANSFERASE (CbiL) FROM METHANOTHERMOBACTER THERMAUTOTROPHICUS^*

Stefanie Frank, Evelyne Deery, Amanda A. Brindley, Helen K. Leech, Andrew Lawrence, Peter Heathcote, Heidi L. Schubert^¶, Keith Brocklehurst, Steve E. J. Rigby, Martin J. Warren¹, and Richard W. Pickersgill²

From the Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom, the School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom, and the ^¶Department of Biochemistry, University of Utah, Salt Lake City, Utah 84112-5650

Ring contraction during cobalamin (vitamin B₁₂) biosynthesis requires a seemingly futile methylation of the C20 position of the tetrapyrrole framework. Along the anaerobic route, this reaction is catalyzed by CbiL, which transfers a methyl group from S-adenosyl-L-methionine to cobalt factor II to generate cobalt factor III. CbiL belongs to the class III methyltransferases and displays similarity to other cobalamin biosynthetic methyltransferases that are responsible for the regiospecific methylation of a number of positions on the tetrapyrrole molecular canvas. In an attempt to understand how CbiL selectively methylates the C20 position, a detailed structure function analysis of the enzyme has been undertaken. In this paper, we demonstrate that the enzyme methylates the C20 position, that its preferred substrate is cobalt factor II, and that the metal ion does not undergo any oxidation change during the course of the reaction. The enzyme was crystallized, and its structure was determined by x-ray crystallography, revealing that the 26-kDa protein has a similar overall topology to other class III enzymes. This helped in the identification of some key amino acid residues (Asp¹⁰⁴, Lys¹⁷⁶, and Tyr²²⁰). Analysis of mutant variants of these groups has allowed us to suggest potential roles that these side chains may play in substrate binding and catalysis. EPR analysis of binary and ternary complexes indicate that the protein donates a fifth ligand to the cobalt ion via a gated mechanism to prevent transfer of the methyl group to water. The chemical logic underpinning the methylation is discussed.

Received for publication, May 9, 2007 , and in revised form, June 13, 2007.

^* The research was supported by Grants BBS/B04145 and BB/E002137 from the Biotechnology and Biological Sciences Research Council. 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.

This work is dedicated to the memory of Prof. Ian Scott.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

The atomic coordinates and structure factors (code 2QBU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence may be addressed. Tel.: 44-1227-824690; E-mail: m.j.warren{at}kent.ac.uk. ² To whom correspondence may be addressed. Tel.: 44-20-7882-6360; E-mail: r.w.pickersgill{at}qmul.ac.uk.

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