title: Mechanism of Flavoprotein l-6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues.
abstract: The flavoenzyme 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 kcat/Kmand kredfor (S)-6-hydroxynicotine, respectively, with larger effects on the kcat/Kmvalue for (S)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the kcat/Kmvalue 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 kcat/Kmvalue 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.
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States.
authors.forename: Paul F
authors.identifier: 0000-0002-8486-2024
authors.initials: PF
authors.lastname: Fitzpatrick
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Chadegani
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States.
authors.forename: Shengnan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry and Structural Biology, University of Texas Health Science Center , San Antonio, Texas 78229, United States.
authors.forename: Vi
authors.initials: V
authors.lastname: Dougherty
mesh_terms: D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009538:Nicotine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D050891:Oxidoreductases Acting on CH-NH2 Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: C502317:6-hydroxynicotine; D001426:Bacterial Proteins; D005420:Flavoproteins; D011994:Recombinant Proteins; D012997:Solvents; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D009538:Nicotine; D050891:Oxidoreductases Acting on CH-NH2 Group Donors; D008239:Lysine