Expression and functional characterization of monoamine oxidase from the zebrafish (Danio rerio): Comparisons with human monoamine oxidases A and B

Monoamine Oxidases (MAO) are flavin containing enzymes located in the outer mitochondrial membrane. Mammals, including humans are shown to contain two forms of this enzyme as MAO A and MAO B. However, not all organisms contain two separate genes expressing these enzymes. Recent studies have shown that zebrafish, a popular teleost organism suitable for various pharmacological applications, contains a single MAO gene. It was proposed that human and teleost MAOs are co-orthologs and share a single common ancestor that underwent a gene duplication event. In addition, studies with whole zebrafish neural tissues have shown that zebrafish MAO exhibit properties closer to human MAO A. To test this hypothesis and to provide the first detailed characterization of zebrafish MAO (zMAO), we developed a high-level expression and purification system for zMAO where we could obtain 235 mg of protein from 0.5 L culture of Pichia pastoris. Then we performed the first detailed functional analysis of the protein with various MAO A and MAO B specific substrates and inhibitors. Here, we also present a comprehensive analysis of quantitive structure relationship of zMAO catalysis in comparison with the human MAO isoforms. Overall data suggest that zMAO contains the properties of both human MAO A and MAO B with properties closer to those of MAO A. The studies from this dissertation provide extensive analysis of this single form of the enzyme and are aimed to be helpful in the pharmacological studies that target designing better drugs targeting MAO using this zebrafish as a system.