EFFECT OF METAL ON 2,4,5-TRIHYDROXYPHENYLALANINE (TOPA) QUINONE BIOGENESIS IN THE HANSENULA-POLYMORPHA COPPER AMINE OXIDASE

Previous studies of wild-type and mutant forms of a recombinant copper amine oxidase from Hansenula polymorpha, expressed in Saccharomyces c erevisiae, have indicated a self-processing mechanism for 2,4,5-trihyd roxyphenylalanine (topa) quinone biogenesis involving the active site copper (Cai, D., and Klinman, J. P. (1994) J. Biol. Chem. 269, 32039-3 2042), In contrast to prokaryotic copper amine oxidases, however, it h as not been possible to initiate topa quinone formation by the additio n of exogenous copper to precursor H. polymorpha amine oxidase lacking copper, Metal analysis of copper-depleted wild-type enzyme reveals 0. 2-0.3 mol copper, together with 0.6 mol zinc, Despite changes in the z inc and copper levels in growth media, the level of zinc in purified e nzyme remains fairly constant, Further, we have been unable to displac e protein-bound zinc by exogenously added copper, The H. polymorpha am ine oxidase gene was subsequently expressed in Escherichia coli and fo und to be almost completely free of copper and zinc. In vitro reconsti tution of this apoprotein confirms that zinc binds to H. polymorpha am ine oxidase and prevents reconstitution with copper, By contrast, addi tion of copper first to apoprotein leads to formation of topa quinone and stable activity in the presence of added zinc, These findings indi cate efficient binding of either zinc or copper to a site that undergo es little or no exchange, The data confirm that topa quinone biogenesi s in the H. polymorpha system is catalyzed by copper and occurs in the absence of added factors, We conclude that the mechanisms of cofactor biogenesis in pro- and eukaryotic systems are likely to be similar or identical, The results described herein imply different pathways for the in vivo assembly of heterologously expressed amine oxidases in S. cerevisiae and E. coil.