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  2. Understanding Substrate Selectivity of Phoslactomycin Polyketide Synthase by Using Reconstituted in Vitro Systems

Understanding Substrate Selectivity of Phoslactomycin Polyketide Synthase by Using Reconstituted in Vitro Systems

  • Chembiochem. 2020 Jul 16;21(14):2080-2085. doi: 10.1002/cbic.202000112.
Kyra Geyer 1 Srividhya Sundaram 1 Peter Sušnik 2 Ulrich Koert 2 Tobias J Erb 1 3
Affiliations

Affiliations

  • 1 Department of Biochemistry and Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, 35043, Marburg, Germany.
  • 2 Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Str. 4, 35032, Marburg, Germany.
  • 3 LOEWE Center for Synthetic Microbiology (Synmikro), Karl-von-Frisch-Str. 16, 35043, Marburg, Germany.
Abstract

Polyketide synthases (PKSs) use simple extender units to synthesize complex Natural Products. A fundamental question is how different extender units are site-specifically incorporated into the growing polyketide. Here we established phoslactomycin (Pn) PKS, which incorporates malonyl- and ethylmalonyl-CoA, as an in vitro model to study substrate specificity. We combined up to six Pn PKS modules with different termination sites for the controlled release of tetra-, penta- and hexaketides, and challenged these systems with up to seven different extender units in competitive assays to test for the specificity of Pn modules. While malonyl-CoA modules of Pn PKS exclusively accept their natural substrate, the ethylmalonyl-CoA module PnC tolerates different α-substituted derivatives, but discriminates against malonyl-CoA. We show that the ratio of extender transacylation to hydrolysis controls incorporation in PnC, thus explaining site-specific selectivity and promiscuity in the natural context of Pn PKS.

Keywords

acyltransferases; enzymes; natural products; phoslactomycin; polyketides.

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