Modular polyketide synthases and cis double bond formation: establishment of activated cis-3-cyclohexylpropenoic acid as the diketide intermediate in phoslactomycin biosynthesis.

The majority of modular polyketide synthase (PKS) systems which generate unsaturated products do so with 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 and three double bonds. There is substantial evidence that 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 double bond there is no experimental evidence to determine if they do so directly, or if they also form a 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 -3-cyclohexylpropenoic acid is the diketide intermediate which passes from module 1 to module 2 of the PLM PKS. The 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 C–C double bond of PLM B, but that the subsequent modules of the PKS clearly discriminate between the and -diketide intermediate and do not contain domains capable of catalyzing double bond isomerization.