Abstract
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.
Highlights
Unlike the EutT and PudO enzymes, CobA has broad specificity for its corrinoid substrate, allowing it to adenosylate de novo corrin ring intermediates as well as complete (e.g. cobalamin (Cbl)) and incomplete corrinoids lacking the nucleotide loop (e.g. cobinamide (Cbi)) [4, 16]
In this hypothetical CobA1⁄7FldA complex, the flavin mononucleotide (FMN) cofactor of flavodoxin A (FldA) was within 10 Å of the Cbl substrate in the CobA active site, and hydrophobic patches on both proteins were brought together (Fig. 2D)
We hypothesized that a peptide mimic of this region should block CobA1⁄7FldA interactions
Summary
Unlike the EutT and PudO enzymes, CobA has broad specificity for its corrinoid substrate, allowing it to adenosylate de novo corrin ring intermediates as well as complete (e.g. cobalamin (Cbl)) and incomplete corrinoids lacking the nucleotide loop (e.g. cobinamide (Cbi)) [4, 16]. In vitro the Co(I) ion is generated by the transfer of an electron from reduced flavodoxin A (FldA) to Co(II), an event that is currently thought to occur in the active site of CobA [14]. One unanswered question regarding the mechanism of catalysis by CobA is how the redox potential of the Co(II) to Co(I) transition (Ϫ610 mV) [23] is increased enough so the electron transfer from reduced FldA (Ϫ450 mV; semiquinonine/hydroquinone) [21, 24] can occur. We previously hypothesized that FldA reduces Co(II) to Co(I) in the CobA active site, triggering the attack of the Co(I) nucleophile on the 5Ј carbon of the ribosyl moiety of ATP [14]. We report results of studies aimed at advancing our understanding of CobA1⁄7FldA interactions highlighting the use of computer modeling to generate experimentally testable models
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