Abstract
Bordetella species are known to catabolize nicotinate to fumarate. In an effort to better understand the mechanism of decarboxylation and subsequent hydroxylation of 6‐hydroxynicotinate (6‐HNA) to 2,5‐dihydroxypyridine (2,5‐DHP), the second step in the conversion of nicotinate to fumarate, the gene coding the putative enzyme (NicC) has been characterized. Analysis of the products of the enzymatic reaction by HPLC‐UV and LCMS are consistent with 2,5‐DHP as the expected product. Steady‐state kinetic analysis of the enzyme activity by isothermal titration calorimetry provides a kcat = 29 +/− 1 s−1, a KM (6‐HNA) = 447 +/− 52 μM and a KM (NADH) = 11 +/− 1 μM. Analysis of the binding of 6‐HNA by following the quenching of the protein's inherent fluorescence by titration yields a KD = 6 +/− 1 μM. The substrate analogue, 6‐ chloronicotinate, known to modulate B. pertussis virulence, is shown to be a non‐competitive inhibitor of NicC. Kinetic activity and thermodynamic binding data will be reported for substrate analogues of NicC in an effort to better understand the structural and electronic determinants of substrate specificity.
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