Investigating Substrate Specificity For 6‐Hydroxynicotinate 3‐Monooxygenase (NicC) With Coumalic Acid: Consequences To Catalysis Of Replacing or Removing The Ring Nitrogen

Abstract

6‐Hydroxynicotinate 3‐monooxygenase (NicC) is a group A flavin dependent monooxygenase (FDM) that catalyzes the regioselective decarboxylative hydroxylation of 6‐HNA into 2,5‐dihydroxypyridine (DHP) with the concomitant oxidation of NADH to NAD+. This enzyme‐catalyzed reaction involves three segments: (1) substrate binding, (2) FAD reduction by NADH, and (3) hydroxylation‐decarboxylation of 6‐HNA through a hydroperoxy‐flavin intermediate. Past studies have shown that NicC can catalyze this reaction with alternate aromatic substrates including 4‐hydroxybenzoic acid (4‐HBA), a substrate analogue with a homocyclic ring (i.e. lacking the ring nitrogen). However, 4‐HBA is bound weakly (355 ± 66 μM) and only 50% of the reaction progresses to substrate hydroxylation, versus hydroperoxy‐flavin degradation to H2O2, suggesting the importance of the ring nitrogen in the coupling efficiency of NicC. To further explore the role of the ring nitrogen, the efficiency of the reaction with coumalic acid (CA) was determined. The NicC‐CA dissociation constant measured by monitoring the change in absorbance at 450nm (flavin) shows that NicC binds CA (Kd = 5 ± 1 μM) 10x more tightly than 6‐HNA (Kd = 58 ± 12 μM). Binding of CA by NicC is an isothermal process (∆H = 0), indicating that it is driven by a favorable change in entropy. Analysis of the reaction products by Q‐TOF MS indicates that NicC catalyzes the decarboxylative hydroxylation of CA into 5‐hydroxy‐2H‐pyrane‐2‐one. However, coupling experiments by HPLC analysis and detection of H2O2 by a fluorescence assay both indicate that the reaction with CA is highly uncoupled with only minor 5‐hydroxy‐2H‐pyrane‐2‐one formation. Steady‐state kinetic analyses show that CA bound to NicC enhances its rate of NADH oxidation 16‐fold (by comparison, 6‐HNA enhances the rate of NicC NADH oxidation 30‐fold). Transient state kinetic analyses by stopped flow under anaerobic conditions to permit single‐turnover kinetics shows that, unlike in the binding of 6‐HNA, the binding of CA does not enable the formation of a charge‐transfer complex prior to FAD reduction. These results indicate that an H‐bond accepting group in the aromatic ring at position 1 is important for establishing the correct conformation of the ES complex to enable effective binding, flavin reduction and, ultimately, substrate hydroxylation.