Biosynthesis of 3-methoxy-5-methyl naphthoic acid and its incorporation into the antitumor antibiotic azinomycin BElectronic supplementary information (ESI) available: Bacterial strains and plasmids used in this study (Table S1), characterization of AziB1 as a P450 enzyme (Fig. S1), effect of pH on the relative activity of AziB1 (Fig. S2), time course of AziB1-catalyzed hydroxylation (Fig. S3), characterization of AziB2 as a SAM-dependent enzyme (Fig. S4), effect of pH on the relative activity o

• Main Authors: Ding, Wei, Deng, Wei, Tang, Mancheng, Zhang, Qi, Tang, Gongli, Bi, Yurong, Liu, Wen
• Format: Article
• Language: English
• ISSN: 1742-206X; 1742-2051
• DOI: 10.1039/b926358f

Azinomycin B is a potent antitumor antibiotic that features a set of unusual, densely assembled functionalities. Among them, the 3-methoxy-5-methylnaphthoic acid (NPA) moiety provides an important noncovalent association with DNA, and may, therefore, contribute to the specificity of DNA alkylation for biological activity exhibition. We have previously cloned and sequenced the azinomycin B biosynthetic gene cluster, and proposed that four enzymes: AziB, AziB1, AziB2, and AziA1, are involved in the naphthoate moiety formation and incorporation. In this study, we report in vivo and/or in vitro characterizations of the P450 hydroxylase AziB1, the O -methyltransferase AziB2, and the substrate specificity of the non-ribosomal peptide synthetase (NRPS) AziA1, providing insights into the timing of individual steps in the late-stage modification of 5-methyl-NPA synthesized by the iterative type I polyketide synthase AziB. AziB1 catalyzes a regiospecific hydroxylation at the C3 position of the free naphthoic acid 5-methyl-NPA to produce 3-hydroxy-5-methyl-NPA, and the resulting hydroxyl group is subsequently O -methylated by AziB2 to furnish the methoxy functionality. The di-domain NRPS AziA1 specifically incorporates 3-methoxy-5-methyl-NPA via an unusual A domain to initiate the backbone formation of azinomycin B. AziA1 activates several analogues of the natural starter unit, suggesting a potential for production by metabolic engineering of new azinomycin analogues differing in their NPA moieties. Characterizations of the P450 hydroxylase AziB1, the O -methyltransferase AziB2, and the non-ribosomal peptide synthetase AziA1 lead to complete establishment of the pathway for 3-methoxy-5-methyl naphthoic acid biosynthesis and its incorporation into azinomycin B.