期刊
BIOCHEMISTRY
卷 60, 期 4, 页码 259-273出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.0c00855
关键词
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资金
- Biomolecular Pharmacology Program - NIH [T32GM008541]
- NSF [CHE 150848]
- Skaggs Institute for Chemical Biology
The research elucidated the mechanism of NicA2, showing its preference for oxidizing tertiary amines with an efficient reductive half-reaction and very slow oxidative half-reaction, suggesting that the true oxidizing agent is unknown.
In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O-2 as the oxidizing substrate show that NicA2 has a low apparent K-m of 114 nM for (S)-nicotine with a very low apparent turnover number (k(cat) of 0.006 s(-1)). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to k(cat). X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 angstrom resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O-2 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.
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