posted on 2016-02-23, 00:00authored byFrancesca Salvi, Isela Rodriguez, Donald Hamelberg, Giovanni Gadda
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.