Mechanism of 6-hydroxynicotinate 3-monooxygenase, a flavin-dependent decarboxylative hydroxylase involved in aerobic nicotinic acid degradation.

6-Hydroxynicotinate 3-monooxygenase (NicC) is a Group A FAD-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) with concomitant oxidation of NADH in nicotinic acid degradation by aerobic bacteria. Two mechanisms for the decarboxylative hydroxylation half-reaction have been proposed [Hicks, K. et al. (2016) Biochemistry 55, 3432 - 3446]. Results with the Bordetella bronchiseptica RB50 NicC here show that a homocyclic analogue of 6-HNA, 4-hydroxybenzoic acid (4-HBA), is decarboxylated and hydroxylated by NicC with a 420-fold lower catalytic efficiency than is 6-HNA. The 13 ( V/ K), measured with wild-type NicC by isotope ratio mass spectrometry following the natural abundance of 13 C in the CO2 product, is inverse for both 6-HNA (0.9989 ± 0.0002) and 4-HBA (0.9942 ± 0.0004), and becomes negligible (0.9999 ± 0.0004) for 5-chloro-6-HNA, an analogue that is 10-fold more catalytically efficient than 6-HNA. Covalently bound 6-HNA complexes of NicC are not observed by mass spectrometry. Comparative steady-state kinetic and equilibrium binding analyses of active site NicC variants (C202A, H211A, H302A, H47E, Y215F, Y225F) identify Tyr215 and His47 as critical determinants of both 6-HNA binding (( Kd Y215F)/( Kd WT) > 240; ( Kd H47E)/( Kd WT)> 350) and in coupling rates of 2,5-DHP and NAD+ product formation ([2,5-DHP]/[NAD+ ] = 1.00 (WT), 0.005 (Y215F), and 0.07 (H47E)]. Results of these functional analyses accord with an electrophilic aromatic substitution reaction mechanism in which His47-Tyr215 serves as the general base to catalyze substrate hydroxylation and refine the structural model for substrate binding by NicC.