Biosynthesis of 3-methoxy-5-methyl naphthoic acid and its incorporation into the antitumor antibiotic azinomycin B.
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.
Ding
Wei
W
School of Life Science, Lanzhou University, 222 South Tianshui Rd, Lanzhou 730000, China.
Deng
Wei
W
Tang
Mancheng
M
Zhang
Qi
Q
Tang
Gongli
G
Bi
Yurong
Y
Liu
Wen
W
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D001426:Bacterial Proteins; D002264:Carboxylic Acids; D036341:Intercellular Signaling Peptides and Proteins; D009281:Naphthalenes; D010455:Peptides; C051055:azinomycin B; C085312:1-naphthoic acid; D048630:Polyketide Synthases; D003577:Cytochrome P-450 Enzyme System; D008780:Methyltransferases; D010453:Peptide Synthases