Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to Methylmalonyl-CoA Decarboxylase Structure–Function Studies

Abstract

Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl-S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure–function relationships. To address this problem, we have synthesized a panel of methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar K i values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure–function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases.

Mechanism of 3‐Methylglutaconyl CoA Decarboxylase AibA/AibB: Pericyclic Reaction versus Direct Decarboxylation

“…However, it is not clear how this stabilization is achieved. Apart from a long hydrogen bond to the amide NH of Gly134 B (Figure 4), there is no oxyanion hole present at the active site like in other cofactor‐independent decarboxylases [6, 12–14] …”

Structures of LnmK a Bifunctional Acyltransferase/Decarboxylase with Substrate Analogs Reveals Basis for Selectivity and Stereospecificity

“…We previously synthesized the methylmalonyl-CoA analogs, 2-nitropropionyl-CoA, 2-nitropropionyl-oxa(dethia)CoA, 2-nitropropionyl-amino(dethia)CoA, 2-sulfonoatepropionyl-CoA and 2-sulfonatepropionyl-oxa(dethia)-CoA. 11 Protein Expression and Protein Purification. Expression vectors pBS3080 for Streptomyces atroolivaceus LnmK (Uniprot accession Q8GGP1), pBS3095 for LnmK Y62F and pBS3165 for Streptomyces coelicolor methylmalonyl-CoA epimerase (SCO5398, Uniprot accession Q9L2C2) were previously described.…”

“…6,10 The enzymes were expressed and purified based on pre-viously described procedures with His6 tag removal by TEV protease. 6,10,11 The TEV-cleaved samples were purified by a tandem desalting column to Ni-NTA column washed with desalting buffer consisting of 10 mM triethanolamine pH 7.5 and 40 mM Na2SO4 for LnmK used in crystallography and 10 mM Tris-HCl pH 8.0 and 100 mM NaCl for LnmK, LnmK Y62F and epimerase used in enzyme assays. Fractions containing pure protein as analyzed by SDS-PAGE, were concentrated to ~25 mg/mL, frozen in small aliquots using liquid nitrogen, and stored at -80 °C until experimentation.…”

“…We previously synthesized methylmalonyl-CoA analogs bearing methylmalonyl-thioester isosteres in order to gain insight into enzyme-substrate interactions. 11 In the case of E. coli methylmalonyl-CoA decarboxylase (MMCD), our methylmalonyl-CoA analogs provided insight into enzyme:substrate interactions leading to a clearer mechanism of catalysis. However, MMCD accepts both epimers of methylmalonyl-CoA, leaving the question of whether our analogs are useful for structure-function studies with stereospecific enzymes, due to the analogs being racemic mixtures, Figure 1.…”

Mechanism of 3‐Methylglutaconyl CoA Decarboxylase AibA/AibB: Pericyclic Reaction versus Direct Decarboxylation

“…Apart from a long hydrogen bond to the amide NH of Gly134 B (Figure 4), there is no oxyanion hole present at the active site like in other cofactorindependent decarboxylases. [6,[12][13][14] It is important to note that the catalytic cycle is not complete at E:P. For that to happen, the product has to be released, the protonation state of Cys56 B has to be restored, and a new substrate has to bind. These steps are not possible to treat with the current approach and were not considered explicitly here.…”