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Synergistic Actions of a Monooxygenase and Cyclases in Aromatic Polyketide Biosynthesis

Type II polyketide synthases (PKSs) are enzymes responsible for the biosynthesis of bacterial aromatic polyketides. 2] The type II minimum PKS—which consists of the b-ketoacyl synthase (KSa), chain length factor (CLF or KSb), and acyl carrier protein (ACP)—synthesizes the nascent polyketide chain by successive Claisen-like decarboxylative condensations. The nascent polyketide chain is then subjected to regioselective reduction and cyclization catalyzed by dedicated ketoreductases (KRs) and cyclases, respectively, to yield an aromatic aglycon. In general, these immediate tailoring enzymes function as ACHTUNGTRENNUNGindependent proteins, a feature that has facilitated the use of heterologous combinations of KRs and cyclases for the biosynthesis of new polyketides. Pradimicin A (Scheme 1) is an aromatic polyketide biosynthesized by Actinomadura hibisca P157–2. It contains a dihydrobenzo[a]naphthacenequinone core, derived from a dodecaketide backbone through five regioselective cyclizations. The carboxyl end of the polyketide at ring E is amidated with d-alanine, while the nonaromatic ring D is substituted with a disaccharide consisting of d-xylopyranose and 4-methylamino-4-deACHTUNGTRENNUNGoxy-d-fucopyranose. Together, these structural elements make pradimicin A an effective viral entry inhibitor and a broadspectrum fungicide. Numerous pradimicin analogues, generated by synthetic, semisynthetic, and strain mutagenesis methods, have been reported. As an example, the more water-soluble derivative BMS-181184 (Scheme 1) exhibited antifungal activity against 167 different fungal strains. Because of the complexity of the pradimicin molecule, however, engineered biosynthesis through manipulation of the pradimicin (pdm) PKS enzymes remains an attractive approach for further development of pradimicin analogues. Pradimicin A is a dihydrobenzo[a]naphthacenequinone that belongs to a family of polyketides including rubromycin, griseorhodin, benastatin, and fredericamycin, all of which are derived from biosynthetic intermediates that contain a pentangular aglycon. The recent sequencing of the benastatin and fredericamycin gene clusters, in conjunction with genetic and biochemical experiments, have provided insights into the biosynthesis of this class of polyketides. However, the regioselective cyclization steps that yield the pentangular core have remained uncharacterized. The cyclization steps are significantly different from those dictating the formation of anthraquinones, anthracyclines, tetracyclines, and angucyclines. In particular, it is unclear how the cyclases can direct the regioselective intramolecular aldol condensations, while preventing the extended, dodecaketide backbone from spontaneous cyclization. Recent sequencing of the 39 kb pdm gene cluster from A. hibisca revealed 28 open reading frames (ORFs). Enzymes that are putatively associated with the biosynthesis of the pradimicin aglycon 5, including the previously identified minimal PKS and tailoring enzymes, are shown in Table 1. Consistently with the gene clusters reported for other pentangular polyketides, the pdm gene cluster contains only three putative cyclases (PdmD, PdmK, and PdmL). Using the Streptomyces coelicolor strain CH999 as the heterologous host, we had previously confirmed the Scheme 1. Pradimicins and structurally related aromatic polyketides.