Structure and Mechanism of 6‐Hydroxynicotinate 3‐Monooxygenase (NicC)

Abstract

NicC catalyzes the decarboxylation and oxidation of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine (DHP) in the aerobic degradation of niacin to fumarate in both soil and pathogenic bacteria. The crystal structure of the Pseudomonas putida NicC apoenzyme has been solved to 2.2 Å resolution using SAD phasing. Steady‐state kinetic analysis of NicC activity fit the sequential ternary kinetic model, with inhibition profiles by NAD and DHP supporting a random bi bi mechanism. Dependence of kcat and kcat/KM on pH show critical roles of both general base and acid catalysis, with pKa values of 6.3 and 8.0, respectively. NicC catalytic turnover drops 60% upon incubation with either iodoacetamide or MMTS, suggesting a role for cysteine that will be examined further by analyzing the kinetic consequences of a NicC C201A mutation. The high catalytic efficiencies of NicC with 3‐chloro‐4‐hydroxybenzoic acid (kcat/KM = 1.5 x 105 M‐1s‐1), and 5‐chloro‐6‐hydroxynicotinate (1.4 x 105 M‐1s‐1), compared to that with 6HNA (kcat/KM = 5.5 x 104 M‐1s‐1), negates mechanistic proposals requiring tautomerization at C‐6. The 13V/K kinetic isotope effect is inverse (0.9986 ± 0.0002), supporting a change in hybridization at C‐3 from sp2 to sp3 prior to decarboxylation. The observed lack of irreversible inhibition by 6‐hydroxynicotinaldehyde negates mechanistic proposals requiring the covalent addition of cysteine at C‐3, suggesting a mechanism in which the –OH group from flavin hydroperoxide is added prior to decarboxylation.