Expression of zebrafish (Danio rerio) monoamine oxidase (MAO) in Pichia pastoris: purification and comparison with human MAO A and MAO B

Protein Expr Purif. 2010 Apr;70(2):290-7. doi: 10.1016/j.pep.2010.01.005. Epub 2010 Jan 14.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Benzylamines / metabolism
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Hot Temperature
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Monoamine Oxidase / biosynthesis*
  • Monoamine Oxidase / isolation & purification
  • Monoamine Oxidase / metabolism
  • Monoamine Oxidase Inhibitors / pharmacology
  • Phenethylamines / metabolism
  • Pichia / metabolism
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Zebrafish

Substances

  • Benzylamines
  • Monoamine Oxidase Inhibitors
  • Phenethylamines
  • Recombinant Proteins
  • benzylamine
  • Monoamine Oxidase