Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues

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• 作者：Paul F. FitzpatrickFatemeh Chadegani ; Shengnan Zhang ; Vi Dougherty
• 刊名：Biochemistry
• 出版年：2017
• 出版时间：February 14, 2017
• 年：2017
• 卷：56
• 期：6
• 页码：869-875
• 全文大小：363K
• ISSN：1520-4995

文摘

The flavoenzyme allcaps">l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (S)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon–carbon bond, it has recently been shown to catalyze oxidation of a carbon–nitrogen bond [Fitzpatrick, P. F., et al. (2016) Biochemistry 55, 697–703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in k_cat/K_m and k_red for (S)-6-hydroxynicotine, respectively, with larger effects on the k_cat/K_m value for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the k_cat/K_m value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the k_cat/K_m value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin.