Phenoxazinone Synthase: Mechanism for the Formation of the Phenoxazinone Chromophore of Actinomycin.

摘要

Phenoxazinone synthase is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The mechanism of this complex 6-electron oxidation was determined by using a variety of substituted 2-aminophenols, designed to block the reaction at intermediate stages. Thus, with 3,5-di-tert-butyl-2-aminophenol as substrate, the reaction was blocked at the o-quinone imine, with 5-tert-butyl-2-aminophenol as substrate, the reaction was blocked at the p-quinone imine; and with 5-methyl-2-aminophenol as substrate, the reaction was blocked at the dihydro-2-aminophenoxazinone. These findings suggested a mechanism in which 2-aminophenoxazinone formation proceeded via a quinone imine intermediate that was trapped by a second molecule of 2-aminophenol. Oxidation of the adduct to the p-quinone imine was followed by a second conjugate addition and a final 2-electron oxidation to give the product, 2-aminophenoxazinone. The role of the enzyme in the catalysis of each of these steps was examined. It was found that the second conjugate addition generated a racemic center at C4a, suggesting that this reaction did not occur at the active site. (jes)