Program in Biomolecular Pharmacology, Boston University School of Medicine, 72 East Concord Street, Boston, Massachusetts 02118, United States.
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, United States.
Biochemistry. 2021 Feb 2;60(4):259-273. doi: 10.1021/acs.biochem.0c00855. Epub 2021 Jan 19.
In , the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of ()-nicotine to -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 as the oxidizing substrate show that NicA2 has a low apparent of 114 nM for ()-nicotine with a very low apparent turnover number ( of 0.006 s). 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 ()-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 . X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with ()-nicotine (at 2.7 and 2.3 Å 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 is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.
在本文中，黄素蛋白尼古丁氧化还原酶（NicA2）催化（）-尼古丁氧化为 -甲基-肌氨酸，后者非酶促水解为假氧尼古丁。结构分析揭示了一种单胺氧化酶（MAO）样折叠，具有保守的 FAD 结合域和可变的底物结合域。该黄素酶具有独特的经典芳香笼变体，带有侧翼残基对 W427/N462。以前使用 O 作为氧化底物的机制研究表明，NicA2 对（）-尼古丁的表观 为 114 nM，表观周转率（）非常低（为 0.006 s）。在此，通过瞬态动力学分析了 NicA2 的机制。使用 W427 和 N462 的单一位点变体来探测这些残基的作用。尽管几种变体的氧化酶活性略有提高（7-12 倍），但其使用（）-尼古丁的还原半反应通常明显慢于野生型 NicA2。值得注意的是，野生型 NicA2 的还原半反应比氧化半反应快 5 个数量级，与氧的反应的表观假一级速率常数类似于 。与（）-尼古丁复合物的 N462V 和 N462Y/W427Y 变体的 X 射线晶体结构（分别为 2.7 和 2.3 Å 分辨率）显示没有明显的活性位点重排。在 N462Y/W427Y 中鉴定出第二个底物结合位点，与观察到的底物抑制一致。这些发现共同阐明了一种黄素酶的机制，该酶优先氧化叔胺，具有有效的还原半反应和当 O 是氧化底物时非常缓慢的氧化半反应，这表明真正的氧化剂是未知的。
