Flavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.

Flavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.

Tobacco-soil bacteria have evolved not only to tolerate high concentrations of nicotine, but to degrade it as a primary growth source. The genomes of several of these species have been sequenced, allowing for the identification of unique bacterial degradation pathways. In the Gram-negative bacteria, Pseudomonas putida, the nicotine-degrading gene cluster has been described; the encoded enzymes catabolize nicotine via the pyrrolidine pathway, ultimately forming malate and fumarate. In previous studies, the flavoenzyme, nicotine oxidoreductase (NicA2), has been identified as the first committed step of nicotine catabolism in this organism. Preliminary kinetic analysis reported that NicA2 has high specificity for S-nicotine, but a slow catalytic rate. Taking advantage of its unique evolutionary adaptation, we aim to refine the inherent catalytic function and structural features of NicA2 towards the development of a biotherapeutic for nicotine addiction, nicotine poisoning and tools for nicotine biosensor development. Our goal is to identify the factors contributing to the mechanistic and substrate-binding properties of NicA2 to improve its biotherapeutic potential. This work presents the first crystal structure of NicA2, resolved to 2.2 Å resolution, establishing it as a member of the flavin-dependent amine oxidase family with a conserved amine oxidase fold. Structural analysis identified a unique composition of the canonical aromatic cage (W427 and N462), which flanks the flavin isoalloxazine ring. Additionally, the X-ray crystallographic structure of the NicA2/S-nicotine complex was refined to 2.6 Å resolution, revealing a hydrophobic active site in support of a hydride-transfer mechanism. Analysis of enzyme activity with a series of substrate analogs and kinetic analysis of active-site residues reveal the determinants of substrate binding affording the remarkable specificity of this enzyme. Using site-directed mutagenesis of aromatic cage residues, along with analysis of the kinetics of the reductive and oxidative steps, we demonstrate that the rate-limiting reaction step is in the oxidative half-reaction. Structural analysis of an active-site variant revealed a secondary binding site consistent with kinetic analysis demonstrating substrate inhibition. Together, our findings provide kinetic and structural evidence for the catalytic mechanism of NicA2, expanding the possibilities for the generation of catalytically-efficient variants and supporting its role as a promising therapeutic strategy. / 2021-01-30T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18599号 / 農博第2086号 / 新制||農||1027(附属図書館) / 学位論文||H26||N4874(農学部図書室) / 31499 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM