Polyketides are a diverse class of natural products having important clinical properties, including antibiotic, immunosuppressive and anticancer activities. They are biosynthesized by polyketide synthases (PKSs), which are modular, multienzyme complexes that sequentially condense simple carboxylic acid derivatives. The final reaction in many PKSs involves thioesterase-catalyzed cyclization of linear chain elongation intermediates. As the substrate in PKSs is presented by a tethered acyl carrier protein, introduction of substrate by diffusion is problematic, and no substrate-bound type I PKS domain structure has been reported so far. We describe the chemical synthesis of polyketide-based affinity labels that covalently modify the active site serine of excised pikromycin thioesterase from Streptomyces venezuelae. Crystal structures reported here of the affinity label-pikromycin thioesterase adducts provide important mechanistic insights. These results suggest that affinity labels can be valuable tools for understanding the mechanisms of individual steps within multifunctional PKSs and for directing rational engineering of PKS domains for combinatorial biosynthesis.
Bibliographical noteFunding Information:
This research was generously supported by grants from the University of Minnesota Graduate School (to R.A.F.) and from the US National Institutes of Health (DK042303 to J.L.S. and GM076477 to D.H.S.). J.D.K. was supported by the Hans and Ella McCollum Vahlteich Research Fund at the University of Michigan College of Pharmacy and by an NRSA postdoctoral fellowship from the US National Institutes of Health (F32 GM075641). The authors thank J. Konwerski for expert assistance with protein purification and crystallization and S. Grüschow for expert mass spectrometric analysis. The GM/CA CAT facility at the Advanced Photon Source is supported by the US National Cancer Institute and the US National Institute of General Medical Sciences. Use of the Advanced Photon Source was supported by the US Department of Energy.