Investigating putative key catalytic residues and uncoupled hydroperoxyflavin formation in the mechanism of 6‐hydroxynicotinate‐3‐monooxygenase, a decarboxylative‐hydroxylase in bacterial nicotinate catabolism

Abstract

Agrochemicals, pharmaceuticals and personal care products (APPCPs) are contaminants of emerging concern that are ineffectively remediated by current methodologies. Many APPCPs contain N‐heterocyclic aromatic constituents (NHACs) that are persistent contaminants in aquatic environments and cause adverse effects in non‐target organisms. Understanding bacterial degradation of NHACs may assist future bioremediation techniques. Aerobic bacterial catabolism of nicotinic acid (NA) serves as a model for understanding the biodegradation of NHACs, and a key enzyme of this pathway is 6‐hydroxynicotinate 3‐monooxygenase (NicC). We recently published the first crystal structure and two postulated mechanisms for NicC. Both mechanisms involve acid‐base catalysis, in which active site residue His211 may serve as a key H‐bond donor. Additionally, the Cys202 active site residue may donate a thiol group, playing a role in one postulated mechanism. Kinetic characterization of the H211A and C202A variants shows that the apparent substrate affinity and catalytic turnover are largely unaffected by mutagenesis of these bases (H211A: KM of 6HNA increases 2.1–fold, KM of NADH increases 2.3–fold, kcat increases 30% compared to WT; C202A: KM of 6HNA increases 2.4–fold, kcat drops 15‐fold). These data suggest that the His211 and Cys202 residues are not critical for catalysis. 6‐Hydroxynicotinaldehyde (6‐HNAH), a competitive inhibitor of NicC with respect to 6‐HNA, exhibits tight binding (KD of 6HNAH = 36 ± 2 μM) compared to 6HNA (KD of 6HNA = 190 ± 25 μM), but shows weak inhibition (Ki of 6HNAH = 8.5 ± 2.4 mM). These results are consistent with the possibility that the formation of hydroperoxyflavin (FADHOOH) is uncoupled from the oxidative decarboxylation reaction, and subsequently produces hydrogen peroxide. Hydrogen peroxide production was detected for reactions of WT+6HNAH and H211A+6HNAH, but not detected for WT+6HNA and H211A+6HNA, suggesting that 6HNAH causes uncoupling and His211 is not a key catalytic residue in the mechanism of NicC.Support or Funding InformationCollege of Wooster Sophomore Research ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.