Exploring The Promiscuity Potential Of 6‐Hydroxynicotinate‐3‐Monooxygenase: Consequences To Catalysis Of Adding A Nitrogen At C5 Within The Substrate’s Aromatic Ring

Abstract

Bacterial degradation of nicotinic acid is a model for exploring the bioremediation potential of soil bacteria. One enzyme within this catabolic pathway is 6‐hydroxynicotinate‐3‐monooxygenase (NicC), a group A flavoenzyme that catalyzes the conversion of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine. This decarboxylative hydroxylation reaction is relatively unique in biology. We recently published a study highlighting NicC’s ability to catalyze the decarboxylation and hydroxylation reaction with alternative substrates including 4‐hydroxybenzoate (4HBA) and 5‐chloro‐6‐hydroxynicotinate (5‐Cl‐6HNA). To further explore the structural basis of a substrate that enables tight binding and catalysis with NicC, 2‐hydroxypyrimidine‐5‐caboxylate (2HM5A) was characterized. This 6‐HNA analogue with an extra N atom at C5 was chosen due to its fundamental symmetry and comparable electron distribution as 5‐Cl‐6HNA. Analyses of NicC with 2HM5A show that the expected product, 2,5‐dihydroxypyrimidine (isouracil), is generated, with H2O2 occurring at a mol fraction of about 0.07, indicating that uncoupling between NADH oxidation and substrate hydroxylation is minimal. Equilibrium titration of NicC with 2HM5A by changes in A‐450 nm measure the Kd = 39 ± 6 µM, indicating that this substrate is bound more tightly than 6‐HNA (Kd= 58 ± 12 µM) but less tightly than 5‐Cl‐6‐HNA (Kd= 7 ± 2 µM). Interestingly, the overall ΔA450 elicited by the binding of 2HM5A (0.018) was smaller in comparison to the ΔA450 observed for the binding of 5‐Cl‐6HNA (0.07) or 6‐HNA (0.05). Attenuated flavin movement due the extra nitrogen of the pyrimidine ring in 2HM5A H‐bonding to the isoalloxazine ring may explain this observation. The kinetics of binding 2HM5A by NicC measured by stopped flow spectrophotometry shows that this analogue binds by a two‐step mechanism, with a nearly ten‐fold higher initial affinity for NicC prior to isomerization (ionization) than 6‐HNA. This added affinity may again be explainable by the added H‐bonding potential of this analogue. In the second step of binding, 2HM5A was observed to have a rate constant similar to 5‐Cl‐6HNA (k2= 220 s‐1), possibly indicating that increased electron density at/near the C5 position accelerates this phase of the binding mechanism. How 2HM5A affects NicC’s mechanism of flavin reduction and oxidation will also be presented. These findings provide further evidence of this monooxygenase’s potential to be engineered to hydroxylate other N‐heterocyclic aromatic compounds in bioremediation.