Computer-assisted docking of flavodoxin with the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein:protein interactions but not for catalysisflavodoxin

doi:10.1074/jbc.M506713200

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Papers In Press, published online ahead of print October 5, 2005
J. Biol. Chem, 10.1074/jbc.M506713200
Submitted on June 21, 2005
Accepted on October 5, 2005

Computer-assisted docking of flavodoxin with the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein:protein interactions but not for catalysisflavodoxin

Nicole R. Buan and Jorge C. Escalante-Semerena

Bacteriology, University of Wisconsin-Madison, Madison, WI 53726-4087

Corresponding Author: escalante{at}bact.wisc.edu

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 (AdoCbl) 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 Arg9 and Arg165 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 Arg9 and Arg165 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.

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