LnmK stereospecifically accepts (2R)-methylmalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure−function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and methylmalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-methylmalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller malonyl-CoA substrate. Indeed, our kinetic analysis reveals malonyl-CoA is accepted at 1% of the rate of methylmalonyl-CoA. This study represents another example of how our nitro-and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme−substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure− function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and β-ketoacylsynthases.
We solved crystal structures of LnmK, which that carries out both acyltransfer and decarboxylation in polyketide synthase pathways. Our structures have substrate analogs bound that reveal interactions that likely occur with the substrate and allow modelling of conformational changes and intermediate states.<br>
We solved crystal structures of LnmK, which that carries out both acyltransfer and decarboxylation in polyketide synthase pathways. Our structures have substrate analogs bound that reveal interactions that likely occur with the substrate and allow modelling of conformational changes and intermediate states.<br>
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.