title: Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase.
abstract: Choline oxidase from Arthrobacter globiformis catalyzes the oxidation of choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of choline oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type choline oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type choline oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of choline oxidase in the oxidative half-reaction. Molecular dynamics studies suggest that the slow isomerization related to F357 is possibly due to the participation of the phenyl ring in a newly proposed gating mechanism for a narrow tunnel, assumed to regulate the access of oxygen to the reduced cofactor.
authors.affiliation: Department of Chemistry, ‡Department of Biology, §Center for Biotechnology and Drug Design, and ∥Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302, United States.
authors.forename: Francesca
authors.initials: F
authors.lastname: Salvi
authors.affiliation: Department of Chemistry, ‡Department of Biology, §Center for Biotechnology and Drug Design, and ∥Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302, United States.
authors.forename: Isela
authors.initials: I
authors.lastname: Rodriguez
authors.affiliation: Department of Chemistry, ‡Department of Biology, §Center for Biotechnology and Drug Design, and ∥Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302, United States.
authors.forename: Donald
authors.initials: D
authors.lastname: Hamelberg
authors.affiliation: Department of Chemistry, ‡Department of Biology, §Center for Biotechnology and Drug Design, and ∥Center for Diagnostics and Therapeutics, Georgia State University , Atlanta, Georgia 30302, United States.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gadda
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001173:Arthrobacter; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation / Q000235:genetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D010100:Oxygen