Biosynthesis of anthracydines: enzymic conversion of aklanonic acid to aklavinone and  -rhodomycinone by anthracycline-producing streptomycetes

Abstract

Aklanonic acid, a substituted anthraquinone, is the earliest stable intermediate isolated so far in the biosynthesis of anthracycline antibiotics. Desalted, soluble cell extracts of Streptomyces sp. C5 and Streptomyces peucetius (ATCC 29050) converted aklanonic acid to E-rhodomycinone in an S-adenosyl-L-methionine-and NADPHdependent sequence of reactions. Aklanonic acid was methylated to form aklanonic acid methyl ester (AAME), which was cyclized to form aklaviketone. Aklaviketone was reduced to aklavinone by an NADPH-linked reductase. When extracts of Streptomyces sp. C5 and S. peucetius were used, aklavinone was hydroxylated at C-11 by an NADPH-and oxygen-dependent reaction to form E-rhodomycinone. Cell extracts of Streptomyces galilaeus strains ATCC 31133 and 31671 converted aklanonic acid to aklavinone in the same manner, but neither of the S. gdilaeus strains hydroxylated aklavinone to E-rhodomycinone. Mutants of Streptomyces sp. C5 blocked at steps in the conversion of aklanonic acid to aklavinone, and which accumulated aklanonic acid (SC5-39; duuC), AAME (SC5-138; duuD, duuE), and aklaviketone (SO-159; duuE, duuF), lacked the expected enzymic activities. In a mutant (SC5-24; duuE) lacking aklaviketone reductase, but retaining aklavinone 11-hydroxylase activity, maggiemycin was formed as an apparent shunt product. An enzymic pathway for 8-rhodomycinone and maggiemycin formation is consistent with the data presented.

Biosynthesis of anthracyclines: analysis of mutants of Streptomyces sp. strain C5 blocked in daunomycin biosynthesis

“…The extract feeding experiments indicated that maggiemycin produced by the dauE mutants is not converted to glycosides, even though the dauE mutant could convert intermediates excreted by other, later mutants, to glycosides. These data, along with experiments showing that maggiemycin cannot be converted to erhodomycinone in vitro (Connors et al, 1990a), demonstrate that maggiemycin is a shunt product of dauE mutants blocked in daunomycin production, rather than an intermediate in the biosynthesis pathway (Fig. 2).…”

“…According to the hypothesis proposed by Eckardt & Wagner (1 988), maggiemycin-accumulating strains are deficient in their ability to convert aklaviketone to aklavinone, which is the normal C-11 hydroxylase substrate. In the absence of the aklavinone reductase, aklaviketone accumulates and is apparently hydroxylated at C-1 1 to produce maggiemycin (Connors et al, 1990a). The extract feeding experiments indicated that maggiemycin produced by the dauE mutants is not converted to glycosides, even though the dauE mutant could convert intermediates excreted by other, later mutants, to glycosides.…”

“…The fact that we isolated double mutants (dauD, E ) that accumulated AAME indicates that the cyclization is enzymecatalysed. Moreover, AAME was the predominant product of the in vitro conversion of aklanonic acid by strain SC5-138, a dauD, E double mutant (Connors et al, 1990a). The reason these mutants are slightly leaky (i.e.…”

“…If this is true, then aklaviketone appears to be a substrate for the formation of both aklavinone by a reduction at C-7 and for maggiemycin by hydroxylation at C-11 ( Fig. 2; Connors et al, 1990a). Thus, the only way in which an aklaviketone accumulating mutant could be obtained would be to have a double mutant (aklaviketone 7-reductase-and C-1 1 hydroxylase-deficient), so that aklavinone would not be produced as an intermediate, and aklaviketone accumulated would not be utilized as a C-1 1 hydroxylase substrate (producing maggiemycin).…”

“…&-Rhodomycinone is formed by hydroxylation of aklavinone at C-1 1 (Connors et al, 1990a). Whereas most early intermediates in the anthracycline biosynthesis pathway are yellow, c-rhodomycinone and other compounds containing the C-1 1 hydroxyl group are red or orange (reviewed by Strohl et al, 1989).…”