Cloning and Sequencing of Phenylethylamine Oxidase from Arthrobacter globiformis and Implication of Tyr-382 as the Precursor to Its Covalently Bound Quinone Cofactor

Abstract

The gene of Arthrobacter globiformis encoding a quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues is a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which has been found to be highly conserved in other copper amine oxidases. Mutation of the former Tyr (Tyr-382) of the recombinant enzyme into Phe resulted in the complete loss of catalytic activity and disappearance of the quinone compound that is specifically detected in the wild-type enzyme, suggesting that Tyr-382 is the precursor to the covalently-bound cofactor, most probably topa quinone. Furthermore, the expression of the active, quinone-containing enzyme in Escherichia coli cells was markedly dependent on the presence of Cu 2+ ions in the culture medium, and the inactive, Cu 2+-deficient enzyme produced without Cu 2+ ions could be converted to the active quinone form by reconstitution with Cu 2+ ions.