Effector Or Substrate: Characterizing Molecular Features of 6‐HNA That Govern Enhanced Rates of NADH Oxidation and Hydroxylation by 6‐Hydroxynicotinate‐3‐Monooxygenase (NicC)

Abstract

Group A flavin‐dependent monooxygenases are involved in the degradation of aromatic compounds and the biosynthesis of natural products. These enzymes catalyze the regioselective incorporation of one oxygen atom from dioxygen into their substrates. Comparison of the mechanisms through which the enzymes in this class work could assist in future bioremediation efforts and aid in lowering the costs of synthesis of hydroxylated aromatic compounds. 6‐Hydroxynicotinate 3‐monooxygenase (NicC), a member of this class of enzymes, catalyzes the decarboxylative‐hydroxylation of 6‐hydroxynicotinic acid (6‐HNA) to 2,5‐dihydroxypyridine with concomitant oxidation of NADH. Like other enzymes in its class, the binding of 6‐HNA stimulates an increase in the rate of oxidation of NADH, however the structural determinants of 6‐HNA responsible for this are unknown. Here we show by kinetic analyses of NicC variants that NicC uses Arg184 to form an ion pair with the carboxylate of 6‐HNA to stabilize the ES complex. Once 6‐HNA is bound, an electronegative atom para to the carboxylate (the 6‐oxo group in 6‐HNA) strongly increases the rate of NADH oxidation, possibly by changing the hydrogen bonding network in the active site and allowing the FAD cofactor to swing to the “out orientation”, allowing for closer contact with NADH and therefore more efficient hydride transfer. Previous work showed that NicC is able to bind its substrates (6‐HNA and 5‐chloro‐6‐HNA) via a 2‐step mechanism, where deprotonation of the pyrindolic group of the substrate governs the second step. Molecules that bind and increase the rate of NADH oxidation without turning over into products are called effectors. Thus, effectors share a common molecular characteristic that prevents them from undergoing the coupled hydroxylative pathway in NicC. Through this we sought to determine the 6‐HNA analogues that act as effectors of NicC in attempt to determine how NicC distinguishes between substrates and effectors. 6‐Chloro‐nicotinic acid, 6‐amino‐nicotinic acid and 6‐methyl nicotinic acid were found to be effectors in NicC (Fig 1). These three molecules are not likely to be ionized by deprotonation, signifying that deprotonation of the substrate could govern the coupled and uncoupled hydroxylation pathways in NicC (Scheme 1). Thus, NicC’s mechanism of controlling NADH reduction of FAD appears to be distinct from the mechanism utilized by p‐hydroxybenzoic acid hydroxylase, the quintessential group A flavin dependent monooxygenase.