Structural and Biochemical Characterization of 6-Hydroxynicotinic Acid 3-Monooxygenase, A Novel Decarboxylative Hydroxylase Involved in Aerobic Nicotinate Degradation
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 kcat/KM6-HNA of 5.0 × 104 M–1 s–1 for B. bronchiseptica NicC. The pH dependence of Vmax/[E]t fits a double-bell model showing an optimum around pH 8 with apparent pKas of 7.24 ± 0.08 and 8.64 ± 0.08, whereas the apparent catalytic efficiency (kcat/KM6-HNA) is maximal around pH 7 and decreases at high pH with an apparent pKa 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 (Ki = 3000 ± 400 μM) in comparison to the apparent binding of 6-HNA (KM = 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.
Hicks, Katherine A.; Yuen, Meigan E.; Zhen, Wei Feng; Gerwig, Tyler J.; Story, Ryan W.; Kopp, Megan C.; and Snider, Mark, "Structural and Biochemical Characterization of 6-Hydroxynicotinic Acid 3-Monooxygenase, A Novel Decarboxylative Hydroxylase Involved in Aerobic Nicotinate Degradation" (2016). Biochemistry, , 3432-3446. 10.1021/acs.biochem.6b00105. Retrieved from https://openworks.wooster.edu/facpub/383