Structural and Biochemical Characterization of 6-Hydroxynicotinic Acid 3-Monooxygenase, A Novel Decarboxylative Hydroxylase Involved in Aerobic Nicotinate Degradation

Abstract

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.

The Bordetella bronchiseptica nic locus encodes a nicotinic acid degradation pathway and the 6‐hydroxynicotinate‐responsive regulator BpsR

“…Recent studies produced recombinant B . bronchiseptica NicF and NicC in Escherichia coli , confirmed and characterized their maleamate amidohydrolase and 6‐hydroxynicotinate 3‐monooxygenase activities respectively, and determined the NicC crystal structure (Kincaid et al ., ; Hicks et al ., ). These findings indicate that the B .…”

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase

“…According to the DALI server [47], the apo NahG model displays good structural agreement with the salicylate hydroxylase from P. putida S-1 (SALH, PDB entry 5EVY) [10], in addition to the homologues urato oxidase from Klebsiella pneumoniae (HpxO, PDB entry 3RP7) [ [22], and flavin-containing monooxygenase from Pseudomonas aeruginosa (PhzS, PDB entry 2RGJ) [55]. The root mean square deviation for these enzymes with respect to NahG is less than 3.2 Å for at least 332 Cα atoms with Z-scores of ≥ 32.4 ( Figure S3).…”

“…The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.to the substrate phenol group [22]. In the reaction catalyzed by 3-hydroxybenzoate 6hydroxylase (3HB6H), which the substrate carboxylate group is meta to the phenol, the 3-hydroxybenzoate is converted to 2,5-dihydroxybenzoate through a hydroxylation followed by a deprotonation [23].Catalysis in these enzymes involves a C(4a)-hydroperoxyflavin species, which provides a powerful peroxo electrophile tuned for oxygen insertion at nucleophilic carbons and soft centers.Examples include hydroxylation coupled with different kinds of substitution, epoxidation, Baeyer-Villiger oxidation, and oxidation of heteroatoms (B, S, Se, N, and P) [24][25][26][27].…”

“…to the substrate phenol group [22]. In the reaction catalyzed by 3-hydroxybenzoate 6hydroxylase (3HB6H), which the substrate carboxylate group is meta to the phenol, the 3-hydroxybenzoate is converted to 2,5-dihydroxybenzoate through a hydroxylation followed by a deprotonation [23].…”

The Novel Monocomponent FAD-dependent Monooxygenase HpaM Catalyzes the 2-Decarboxylative Hydroxylation of 5-Hydroxypicolinic Acid inAlcaligenes faecalisJQ135

“…So far, only two of them (NicC and HspB) catalyzed the 3-decarboxylative hydroxylation of the pyridine-ring (Fig. 5B) (8, 20, 21). To the best of our knowledge, HpaM was the first identified flavin-dependent monooxygenase that catalyzed the 2-decarboxylative hydroxylation of pyridine or pyridine derivatives.…”