Copper ion-dependent biogenesis of topa quinone cofactor covalently bound to bacterial monoamine oxidase

Abstract

The gene of a Gram-positive Coryneform bacterium Arthrobacter globiformis encoding a copper-containing quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues was found a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which is highly conserved in this class of enzymes. The former Tyr (Tyr-382) in the consensus sequence is supposed to be the precursor to the covalentlybound 2,4,5-trihydroxyphenylalanine (topa) quinone cofactor. To elucidate the mechanism of the quinone cofactor formation, an expression plasmid has been constructed for the cloned phenylethylamine oxidase gene, and the recombinant enzyme was overproduced in a Cu2+-depleted medium. The Cu2+-deficient inactive enzyme purified to homogeneity was dramatically activated upon incubation with Cu2+ under aerobic conditions, concomitantly with the formation of the topa quinone at the position corresponding to Tyr-382. The topa quinone formation was accelerated by hydrogen peroxide and retarded by catalase, but required no external enzymatic systems. The purified mutant enzyme, in which the precursor Tyr-382 to the topa quinone is replaced by Phe, had neither activity nor the topa quinone cofactor even after reconstitution with Cu2+. These results demonstrate the Cu2+-dependent autooxidation of a specific tyrosyl residue into the topa quinone cofactor. A possible mechanism of the topa quinone formation is discussed here, which represents a novel type of posttranslational modification of proteins to generate a covalently bound catalytic group.