Phoslactomycin C

Phoslactomycins A to F are new antifungal antibiotics produced by Streptomyces nigrescens SC-273. On the basis of physico-chemical properties and NMR studies, their structures have been determined as shown in Fig. 6. They are characterized by possessing alpha, beta-unsaturated delta-lactone, phosphate ester, conjugated diene and cyclohexane ring moieties. The structural difference between them is ascribed to a substituent bound to the cyclohexane ring.

Polyketide synthases (PKSs) use simple extender units to synthesize complex natural products. A fundamental question is how different extender units are site-specifically incorporated into the growing polyketide. Here we established phoslactomycin (Pn) PKS, which incorporates malonyl- and ethylmalonyl-CoA, as an in vitro model to study substrate specificity. We combined up to six Pn PKS modules with different termination sites for the controlled release of tetra-, penta- and hexaketides, and challenged these systems with up to seven different extender units in competitive assays to test for the specificity of Pn modules. While malonyl-CoA modules of Pn PKS exclusively accept their natural substrate, the ethylmalonyl-CoA module PnC tolerates different α-substituted derivatives, but discriminates against malonyl-CoA. We show that the ratio of extender transacylation to hydrolysis controls incorporation in PnC, thus explaining site-specific selectivity and promiscuity in the natural context of Pn PKS.

Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays. Streptomyces sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of plmS(3) from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp plmS(3) open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in Escherichia coli and shown to be an 18-O acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (k(cat) of 28 +/- 3 min(-1) and K(m) of 88 +/- 16 microM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS(3) was shown to catalyze esterification of PLM G with CoA and N-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.