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

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.

Figure 1

SDS-polyacrylamide gel mobility of purified zMAO A. Coomassie-stained protein bands. Molecular weight marker (lane 1), purified zMAO (lane 2), and human MAO A (lane 3) B. Western blot using antisera specific for covalent flavin coenzymes. Lane 1 is purified zMAO, lane 2 is purified human MAO A. The arrow defines a mobility corresponding to 60 kD.

Figure 1

SDS-polyacrylamide gel mobility of purified zMAO A. Coomassie-stained protein bands. Molecular weight marker (lane 1), purified zMAO (lane 2), and human MAO A (lane 3) B. Western blot using antisera specific for covalent flavin coenzymes. Lane 1 is purified zMAO, lane 2 is purified human MAO A. The arrow defines a mobility corresponding to 60 kD.

Figure 2

A. Absorption spectra of zMAO in its oxidized form (—), after photoreduction to a mixture of its anionic semiquinone and hydroquinone forms (…), and after air reoxidation (----). The sample was dissolved in 50 mM potassium phosphate, 0.5 mM EDTA, pH 7.2 with a 5-deazaflavin concentration of 0.5 μM. B. X-Band EPR spectrum of the photoreduced zMAO sample from 2A. Spectral conditions: 2 mW power, 2 gauss modulation amplitude, temperature = 160°. The spectrum is the average of 200 scans. The arrows denote the maxima and minima corresponding to the peak-to-peak separation referred to in the text.

Figure 2

A. Absorption spectra of zMAO in its oxidized form (—), after photoreduction to a mixture of its anionic semiquinone and hydroquinone forms (…), and after air reoxidation (----). The sample was dissolved in 50 mM potassium phosphate, 0.5 mM EDTA, pH 7.2 with a 5-deazaflavin concentration of 0.5 μM. B. X-Band EPR spectrum of the photoreduced zMAO sample from 2A. Spectral conditions: 2 mW power, 2 gauss modulation amplitude, temperature = 160°. The spectrum is the average of 200 scans. The arrows denote the maxima and minima corresponding to the peak-to-peak separation referred to in the text.

Figure 3

Fluorescence emission spectra of tryptic zMAO flavin peptide before (…) and after performic acid oxidation (—). The emission spectrum of an equal concentration of riboflavin (----) is shown for comparison. An excitation wavelength of 345 nm was used. All samples were dissolved in glass distilled water.

Figure 4

Thermal stability of purified zMAO catalytic activity. The buffer medium is 50 mM potassium phosphate, pH 7.2 containing 0.8% (w/v) OGP. Kinetic assays were measured at 25 °C using kynuramine as substrate as described in the experimental section.

Figure 5

Inhibition of zMAO by Clorgyline and by Deprenyl. A. IC₅₀ values of zMAO for the irreversible inhibitors Clorgyline (—) and Deprenyl (-----); values are 6.4 × 10⁻⁵ M and 6.5×10⁻⁶ M, respectively. Activities were measured at 25 °C using kynuramine as substrate. B. Absorption spectra of purified zMAO before (—) and after the addition of a 10-fold molar excess of Deprenyl (…..) and of Clorgyline (-----). The samples were dissolved in 50 mM potassium phosphate, pH 7.4 containing 0.8% (w/v) OGP.

Figure 5

Inhibition of zMAO by Clorgyline and by Deprenyl. A. IC₅₀ values of zMAO for the irreversible inhibitors Clorgyline (—) and Deprenyl (-----); values are 6.4 × 10⁻⁵ M and 6.5×10⁻⁶ M, respectively. Activities were measured at 25 °C using kynuramine as substrate. B. Absorption spectra of purified zMAO before (—) and after the addition of a 10-fold molar excess of Deprenyl (…..) and of Clorgyline (-----). The samples were dissolved in 50 mM potassium phosphate, pH 7.4 containing 0.8% (w/v) OGP.