The genes coding for the enzymes of oxidative degradation of nicotinic acid have recently been identified in several species of aerobic bacteria, namely, Pseudomonas putida KT2440, Bordetella bronchiseptica RB50, and Bacillus niacini. One of the enzymes involved in an early step of this pathway is a flavin-dependent monooxygenase (6-hydroxynicotinic acid 3-monooxygenase; NicC) that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP), with concomitant oxidation of NADH to NAD+. The nicC genes from B. bronchiseptica RB50 and P. putida have been cloned, and the purified enzymes have been characterized functionally and structurally. Global fits of the steady-state kinetic data show that both enzymes are efficient catalysts, with an apparent k cat/K M 6‑HNA of 5.0 × 104 M–1 s–1 for B. bronchiseptica NicC. The pH dependence of V max/[E] t fits a double-bell model showing an optimum around pH 8 with apparent pK as of 7.24 ± 0.08 and 8.64 ± 0.08, whereas the apparent catalytic efficiency (k cat/K M 6‑HNA) is maximal around pH 7 and decreases at high pH with an apparent pK a of 7.60 ± 0.06. The enzyme’s relative affinity for 6-hydroxynicotinaldehyde, a neutral analogue that shows competitive inhibition with respect to 6-HNA, is weak (K i = 3000 ± 400 μM) in comparison to the apparent binding of 6-HNA (K M = 85 ± 13 μM). The crystal structure for P. putida NicC has been solved to 2.1 Å using SAD phasing, and the 6-HNA substrate has been modeled into the active site. Together these data provide insight into potential reaction mechanisms for this novel decarboxylative hydroxylation reaction.

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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.