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J. Biol. Chem., Vol. 280, Issue 49, 40948-40956, December 9, 2005

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Computer-assisted Docking of Flavodoxin with the ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme Reveals Residues Critical for Protein-Protein Interactions but Not for Catalysis^*

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53726

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.

Received for publication, June 21, 2005 , and in revised form, October 3, 2005.

^* This work was supported in part by National Institutes of Health Grant GM40313 (to J. C. E.-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.

¹ Supported in part by a Howard Hughes Predoctoral Fellowship. To whom correspondence should be addressed: Dept. of Bacteriology, 1710 University Ave., Madison, WI 53726-4087. Tel.: 608-262-7379; Fax: 608-265-7909; E-mail: escalante{at}bact.wisc.edu.

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