Polyketides are a class of biologically active microbial and plant-derived metabolites that possess a high degree of structural and functional diversity and include many human therapeutics, among them anti-infective and anti-cancer drugs, growth promoters and anti-parasitic agents. The macrolide antibiotics, characterized by a glycoside-linked macrolactone, constitute an important class of polyketides, including erythromycin and the natural ketolide anti-infective agent pikromycin. Here we describe new mechanistic details of macrolactone ring formation catalyzed by the pikromycin polyketide synthase thioesterase domain from Streptomyces venezuelae. A pentaketide phosphonate mimic of the final pikromycin linear chain-elongation intermediate was synthesized and shown to be an active site affinity label. The crystal structures of the affinity-labeled enzyme and of a 12-membered-ring macrolactone product complex suggest a mechanism for cyclization in which a hydrophilic barrier in the enzyme and structural restraints of the substrate induce a curled conformation to direct macrolactone ring formation.
Bibliographical noteFunding Information:
This research was generously supported by grants from the University of Minnesota Graduate School (to R.A.F.), by grants DK042303 (to J.L.S.) and GM076477 (to D.H.S.) from the US National Institutes of Health (NIH). J.D.K. was supported by the Hans and Ella McCollum Vahlteich Research Fund at the University of Michigan College of Pharmacy and an NIH postdoctoral fellowship (GM075641). The authors thank L. Venkatraman for the synthesis of aldehyde 15 and J. Konwerski for expert assistance with protein purification and crystallization. The GM/CA Collaborative Access Team facility at the APS is supported by the NIH Institute of General Medical Sciences (GM) and National Cancer Institute (CA). Use of the APS was supported by the US Department of Energy.