EVIDENCE FOR 2 CATALYTICALLY INDEPENDENT CLUSTERS OF ACTIVE-SITES IN A FUNCTIONAL MODULAR POLYKETIDE SYNTHASE

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), catalyze the biosynthesis of structurally complex a nd medicinally important natural products. These large multifunctional enzymes are organized into ''modules'', where each module contains ac tive sites homologous to those of higher eucaryotic fatty acid synthas es (FASs). Like FASs, modular PKSs are known to be dimers, Here we pro vide functional evidence for the existence of two catalytically indepe ndent clusters of active sites within a modular PKS. In three bimodula r derivatives of DEBS, the ketosynthase domain of module 1 (KS-I) or m odule 2 (KS-2) or the acyl carrier protein domain of module 2 (ACP-2) was inactivated via site-directed mutagenesis. As expected, the purifi ed proteins were unable to catalyze polyketide synthesis (although the KS-l mutant could convert a diketide thioester into the predicted tri ketide lactone). Remarkably however, the KS-1/KS-2 and the KS-2/ACP-2 mutant pairs could efficiently complement each other and catalyze poly ketide formation. In contrast, the KS-1 and ACP-2 mutants did not comp lement each other, On the basis of these a:nd other results, a model i s proposed in which the individual modules of a PKS dimer form head-to -tail homodimers, thereby generating two equivalent and independent cl usters of active sites for polyketide biosynthesis. Specifically, each subunit contributes half of the KS and ACP domains in each cluster. A similar complementation approach should also be useful in dissecting the organization of the remaining types of active sites within this fa mily of multienzyme assemblies. Finally, blocked systems, such as the KS-1 mutant described here, present a new strategy for the noncompetit ive conversion of unnatural substrates into polyketides by modular PKS s.