Protein Engineering Studies Toward Development of a GABA Oxidase from an N-Methyl-GABA Oxidase

Abstract

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain, and a method for its detection with high temporal and spatial resolution is highly desirable for many research and clinical applications. One such method is amperometric biosensing, which relies on the activity of a highly substrate-specific peroxide-producing flavin oxidase enzyme with appropriate kinetic parameters. Such an enzyme must have the appropriate kinetic parameters, including kcat, KM, and pH optimum, to facilitate GABA detection in the environment to be monitored. Since there are no known GABA oxidase enzymes that satisfy all of these requirements, we undertook the engineering of an N-methyl-GABA oxidase into a GABA oxidase that could operate at physiological pH. We obtained a partial crystal structure of the enzyme, and used this to develop rational design and site saturation mutagenesis approaches to identify sites for mutagenesis that would enhance the enzyme’s specificity, affinity, and pH optimum with respect to GABA. Much of our problems stemmed from earlier reports on the N-methyl-GABA oxidase enzyme (MAO) which suggested a set of operational parameters which could not be reproduced. Alignment with other amine oxidase class members and docking studies provided conflicting insights as to how to proceed. While we were unable to produce a fully functional GABA oxidase, this work nonetheless provides the first crystallographic model of MAO’s active site, and the identification of those residues within the active site that are not amenable to change. As part of this work, we also correct misleading reports in the literature that might influence other research efforts on MAO. Finally, we provide a framework for the future development of an enzyme suitable for use in a GABA biosensor.