Structures of LnmK, a Bifunctional Acyltransferase/Decarboxylase, with Substrate Analogues Reveal the Basis for Selectivity and Stereospecificity

Abstract

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.

Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases

“…In the structure of MMCD, the sulfonate group is located in a small hydrophobic pocket comprising Pro133, Val138, and Tyr140, and this space is proposed to assist decarboxylation in a similar manner to that proposed above for the GfsA KS Q domain (Figure S16A). LnmK is also proposed to catalyze decarboxylation of methylmalonyl-CoA by fixing the conformation of the substrate and promoting decarboxylation by binding the carboxylate in a hydrophobic pocket . Although the GfsA KS Q domain has no sequence similarity to MMCD or LnmK, these enzymes catalyze decarboxylation in a similar manner.…”

“…LnmK is also proposed to catalyze decarboxylation of methylmalonyl-CoA by fixing the conformation of the substrate and promoting decarboxylation by binding the carboxylate in a hydrophobic pocket. 21 Although the GfsA KS Q domain has no sequence similarity to MMCD or LnmK, these enzymes catalyze decarboxylation in a similar manner.…”

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria

“…These experiments identify the Ile44-Met50 loop as the Mad active site. The methylmalonyl-ACP decarboxylase encoded within the limoline polyketide gene cluster (LmnK) has a double hot dog fold and also uses an asparagine residue on a similar loop to catalyze decarboxylation ( 27 , 28 ).

Figure 4 Structure and catalytic loop of MadB.

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“…The methylmalonyl-ACP decarboxylase encoded within the limoline polyketide gene cluster (LmnK) has a double hot dog fold and also J o u r n a l P r e -p r o o f uses an asparagine residue on a similar loop to catalyze decarboxylation (27,28).…”

Structures of chloramphenicol acetyltransferase III and E. coli β-ketoacylsynthase III co-crystallized with partially hydrolysed acetyl-oxa(dethia)CoA

“…(Jencks & Gilchrist, 1964, Yang & Drueckhammer, 2001 We recently synthesized AcOCoA and similar analogs to support structure-function studies. (Stunkard, Kick, et al, 2021, Stunkard, Benjamin, et al, 2021, Stunkard et al, 2019 However, there was a report of the AcOCoA synthesis before ours. (Weeks et al, 2018) In that study, a similar substrate analog, fluoroacetyl-oxa(dethia)CoA was hydrolyzed 500-fold slower by a thioesterase than the native fluoroacetyl-CoA substrate.…”