PHENOXAZINONE SYNTHASE - MECHANISM FOR THE FORMATION OF THE PHENOXAZINONE CHROMOPHORE OF ACTINOMYCIN
PHENOXAZINONE SYNTHASE - MECHANISM FOR THE FORMATION OF THE PHENOXAZINONE CHROMOPHORE OF ACTINOMYCIN
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DOI：
10.1021/bi00441a026
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发表时间：
1989-07-25
期刊：
影响因子：
2.9
通讯作者：
BEGLEY, TP
中科院分区：
文献类型：
--
作者：
BARRY, CE;NAYAR, PG;BEGLEY, TP
Clifton E. Barry, III, Parmesh G. Nayar, and Tadhg P. Begley* Department of Chemistry, Cornell University, Ithaca, New York Ą4853 Received December 8, 1988; Revised Manuscript Received April 18, 1989 abstract: 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-ZerZ-butyl-2-aminophenol (16) as substrate, the reaction was blocked at the o-quinone imine 17; with 5-Ze/7-butyl-2-aminophenol (19) as substrate, the reaction was blocked at the p-quinone imine 20; and with 5-methyl-2-aminophenol (21) as substrate, the reaction was blocked at the dihydro-2-aminophenoxazinone 22. These findings suggested a mechanism in which 2-aminophenoxazinone formation proceeded via a quinone imine intermediate 4 that was trapped by a second molecule of 2-aminophenol. Oxidation of the adduct 5 tothe/7-quinone imine 6 was followed by a second conjugate addition and a final 2-electron oxidationto 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. A deuterium isotope effect on the cleavage of the C4-H bond of 2-aminophenol suggested that partial dissociation of an in-termediate from the enzyme occurred after the first conjugate addition. It is proposed that 2-amino-phenoxazinone synthesis proceeds via a sequence of three consecutive 2-electron aminophenol oxidations and that the aminophenol moiety is regenerated during the reaction sequence by faciletautomerization reactions. Thus, what initially appears to be an impressively complex mechanism may, in fact, be ingeniously simple.-^ Actinomycin D (2) is a member of an interesting class of natural products in which the yellow-red 2-aminophen-oxazinone chromophore is linked totwo cyclic pentapeptides (Katz, 1967; Hollstein, 1974). These compounds are among the most potent antineoplastic agents known. Their clinical use, however, has been limited to the treatment of choriocarcinoma, Wilms tumors, rhabdomyosarcoma, and Kaposi’s sarcoma due to their high toxicity (Freí, 1974). The mechanism of this cytotoxicity has been extensively studied. It has been shown that actinomycin binds to DNA by intercalation of the phenoxazinone chromophore and that the cyclic pen-tapeptide lactone confers sequence specificity to adjacent GC base pairs. This interaction results in highly specific inhibition of DNA-dependent RNA synthesis (Hollstein, 1974; Gale et al., 1981). In addition to the actinomycins, both xanthommatin and cinnabarin havebeen found to contain the phenoxazinone chromophore (Butenandt, 1957; Cavill et al., 1959). The biosynthesis of actinomycin involves the conversion of tryptophan to 3-hydroxyanthranilic acid in a multistep se-quence (Katz, 1967). The pentapeptide lactone is then attached (Keller, 1984) and the resulting 2-aminophenol (1) undergoes a 6-electron oxidative coupling to form actinomycin (eq 1). The latter reaction is catalyzed by phenoxazinone
