Biochemistry, Department of


Date of this Version



The Journal of Biological Chemistry, VOL. 280, NO. 49, pp. 40948–40956, December 9, 2005


Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.


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

CobAand FldA interact, a computer model of aCobA-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.