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 agents¹. 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.

1. Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, USA
2. Department of Medicinal Chemistry, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, USA.
3. Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street S.E., 8-101 Weaver–Densford Hall, Minneapolis, Minnesota 55455-0353, USA.
4. Department of Microbiology & Immunology, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, USA.
5. Department of Chemistry, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, USA.
6. Department of Biological Chemistry, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216, USA.
7. These authors contributed equally to this work.

Correspondence to: Janet L Smith^1,6 Email: JanetSmith@umich.edu

Correspondence to: Robert A Fecik³ Email: fecik001@umn.edu

Correspondence to: David H Sherman^1,2,4,5 Email: davidhs@umich.edu

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