Mechanism of the Flavoprotein l-Hydroxynicotine Oxidase: Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues

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• 作者：Paul F. Fitzpatrick ; Fatemeh Chadegani ; Shengnan Zhang ; Kenneth M. Roberts ; Cynthia S. Hinck
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：February 2, 2016
• 年：2016
• 卷：55
• 期：4
• 页码：697-703
• 全文大小：344K
• ISSN：1520-4995

文摘

The flavoprotein l-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon–carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine [Kachalova, G., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 4800–4805]. Analysis of the product of the enzyme from Arthrobacter nicotinovorans by nuclear magnetic resonance and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon–nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. On the basis of the k_cat/K_m and k_red values for (S)-hydroxynicotine and several analogues, the methyl group contributes only marginally (～0.5 kcal/mol) to transition-state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ～4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.