Copper amine oxidase: heterologous expression, purification, and characterization of an active enzyme in Saccharomyces cerevisiae.
A copper amine oxidase gene from a methylotrophic yeast Hansenula polymorpha has been expressed in Saccharomyces cerevisiae under the control of the ADHI promoter and the recombinant protein purified to near homogeneity. The recombinant enzyme is as active as the native enzyme in catalyzing methylamine oxidation. We demonstrate that it is a quinoprotein by redox-cycling staining and titrations with carbonyl reagents. The absorption spectral properties of the recombinant amine oxidase and its phenylhydrazine derivative are very similar to those of other copper amine oxidases. The cofactor in the enzyme is 2,4,5-trihydroxyphenylalanine (topa) quinone, as demonstrated by the pH-dependent shift in the lambda max of the p-nitrophenylhydrazone adduct. Alignment of an active-site peptide and DNA-derived protein sequences reveals a tyrosine residue as the precursor to topa quinone, consistent with findings with other copper amine oxidases. All evidence presented herein indicates that the heterologously expressed copper amine oxidase protein is processed posttranslationally in S. cerevisiae to form an active enzyme with an intact cofactor. This occurs despite an inability of S. cerevisiae to utilize amines as a nitrogen source. The implications of this study for the mechanism of topa quinone biogenesis are discussed.
Department of Chemistry, University of California, Berkeley 94720.
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Klinman
D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001596:Benzylamines / Q000378:metabolism; D001665:Binding Sites; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013053:Spectrophotometry; D013379:Substrate Specificity
D001596:Benzylamines; D008744:Methylamines; D010659:Phenylhydrazines; D011994:Recombinant Proteins; C030299:phenylhydrazine; C030796:benzylamine; C027451:methylamine; D006631:Amine Oxidase (Copper-Containing)