Expression of zebrafish (Danio rerio) monoamine oxidase (MAO) in Pichia pastoris: purification and comparison with human MAO A and MAO B.
The expression, purification and characterization of zebrafish monoamine oxidase (zMAO) using the methylotropic yeast Pichia pastoris expression system is described. A 1L fermentation culture of Pichia pastoris containing the gene encoding zMAO under control of the methanol oxidase promotor expresses approximately 200mg of zMAO exhibiting 300 U of total activity. The enzyme is found in the mitochondrial fraction of the expression host and is purified in a 30% yield as a homogenous species with a M(r) of approximately 60,000 on SDS-PAGE and a mass of 58,525+/-40 Da from MALDI-TOF measurements. The zMAO preparation contains one mole of covalent flavin cofactor per mole of enzyme and exhibits >80% functionality. The covalent flavin exhibits fluorescence and EPR spectral properties consistent with known properties of 8 alpha-S-cysteinyl FAD. Chemical degradation of the flavin peptide results in the liberation of FAD. zMAO exhibits no immuno-chemical cross-reactivity with polyclonal anti-sera raised against human MAO A. The enzyme preparation exhibits reasonable thermostability up to a temperature of 30 degrees C. Benzylamine is oxidized with a k(cat) value of 4.7+/-0.1 min(-1) (K(m)=82+/-9 microM) and the enzyme oxidizes phenylethylamine with a k(cat) value of 204 min(-1) (K(m)=86+/-13 microM). The K(m) (O(2)) values determined for zMAO using either benzylamine or phenylethylamine as substrates ranges from 108(+/-5) to 140(+/-21)microM. The functional behavior of this teleost MAO relative to human MAO A and MAO B is discussed.
Arslan
Betül Kacar
BK
Department of Biochemistry, Emory University, Rollins Research Bldg., 1510 Clifton Road, Atlanta, GA 30322, USA.
Edmondson
Dale E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001596:Benzylamines / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D010627:Phenethylamines / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015027:Zebrafish
D001596:Benzylamines; D008996:Monoamine Oxidase Inhibitors; D010627:Phenethylamines; D011994:Recombinant Proteins; C030796:benzylamine; D008995:Monoamine Oxidase