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Understanding Substrate Selectivity of Phoslactomycin Polyketide Synthase by Using Reconstituted in Vitro Systems
Author(s) -
Geyer Kyra,
Sundaram Srividhya,
Sušnik Peter,
Koert Ulrich,
Erb Tobias J.
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000112
Subject(s) - polyketide , extender , polyketide synthase , stereochemistry , context (archaeology) , substrate (aquarium) , chemistry , acyltransferases , in vitro , biochemistry , combinatorial chemistry , enzyme , biology , biosynthesis , organic chemistry , paleontology , ecology , polyurethane
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.