6X7O

LnmK in complex with 2-sulfonate-propionyl-CoA

Summary for 6X7O

• Entry DOI: 10.2210/pdb6x7o/pdb
• Descriptor: Bifunctional methylmalonyl-CoA:ACP acyltransferase/decarboxylase, (2S)-sulfonatepropionyl-CoA, (2R)-sulfonatepropionyl-CoA, ... (5 entities in total)
• Functional Keywords: transferase, lyase, inhibitor, isostere, transferase-transferase inhibitor complex, transferase/transferase inhibitor
• Biological source: Streptomyces atroolivaceus
• Total number of polymer chains: 1
• Total formula weight: 37571.96

Authors

Stunkard, L.M.,Kick, B.J.,Lohman, J.R. (deposition date: 2020-05-30, release date: 2020-06-10, Last modification date: 2023-10-18)

Primary citation

Stunkard, L.M.,Kick, B.J.,Lohman, J.R.
Structures of LnmK, a Bifunctional Acyltransferase/Decarboxylase, with Substrate Analogues Reveal the Basis for Selectivity and Stereospecificity.
Biochemistry, 60:365-372, 2021

PubMed Abstract: LnmK stereospecifically accepts (2)-methylmalonyl-CoA, generating propionyl--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 (2)-methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl--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 (2)-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.

PubMed: 33482062
DOI: 10.1021/acs.biochem.0c00893

Experimental method

X-RAY DIFFRACTION (1.55 Å)