Modular Polyketide Synthases and <i>cis</i> Double Bond Formation:  Establishment of Activated <i>cis</i>-3-Cyclohexylpropenoic Acid as the Diketide Intermediate in Phoslactomycin Biosynthesis

Abstract

The majority of modular polyketide synthase (PKS) systems which generate unsaturated products do so with trans double bonds. Phoslactomycin B (PLM B) presents a class of antitumor and antiviral natural polyketide products that have unique structural features, including a linear unsaturated backbone with one trans and three cis double bonds. There is substantial evidence that trans double bonds are established by ketoreductase−dehydratase (KR−DH) didomains within a PKS module. In cases where modules containing these didomains appear to generate product containing a cis double bond, there is no experimental evidence to determine if they do so directly or if they also form a trans double bond with a subsequent isomerization step. A critical step in addressing this issue is establishing the stereochemistry of the polyketide intermediate which passes to the subsequent module. Herein, we demonstrate through a series of experiments that an activated cis- 3-cyclohexylpropenoic acid is the diketide intermediate which passes from module 1 to module 2 of the PLM PKS. The trans isomer of the diketide intermediate could not be processed directly into PLM B by module 2 but could be converted to PLM B by degradation to cyclohexanecarboxylic acid and elongation by the entire PLM PKS. These observations indicate not only that module 1 with a DH−KR didomain is responsible for establishing the C 14 −C 15 cis double bond of PLM B but also that the subsequent modules of the PKS clearly discriminate between the cis - and trans -diketide intermediate and do not contain domains capable of catalyzing double bond isomerization.