Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

Abstract

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2'-chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semirational design of the enzyme showed that the W187H variant could increase the activity by ∼100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of the substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. On the basis of these computational results, we further predicted that the mutations W187Y and D287G might also be able to increase the substrate binding and thus improve the enzyme's catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100-fold increase in turnover rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turnover rate. Our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase rPPEs, which will help guide future development of more efficient rPPE variants.

Functional analysis of four upregulated carboxylesterase genes associated with fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval)

“…Many environmental xenobiotics are metabolized because CarE has the ability to hydrolyze the carboxylic esters of these toxic chemicals to non-toxic alcohols and acids. [11][12][13][14] Pyrethroids, known for their rapid knockdown effect against pests at a minimal dose, are detoxified by CarEs leading to rapid resistance accumulation. 15 Two crucial mechanisms confer pyrethroid resistance mediated by CarE genes: elevation of transcription and pointed mutation (G137D and W251L).…”

Identification and characterization of a novel carboxylesterase from Phaseolus vulgaris for detection of organophosphate and carbamates pesticides

“…Partial proteins of a high MOWSE score are listed in Table . Carboxylesterases belong to the α / β hydrolase family and had a relatively conserved protein domain . According to peptide chain sequence, the protein (XP_007149983) containing the α / β hydrolase family conserved domain is found in 108 proteins.…”

“…Carboxylesterases belong to the / hydrolase family and had a relatively conserved protein domain. 34,35 According to peptide chain sequence, the protein (XP_007149983) containing the / hydrolase family conserved domain is found in 108 proteins. The molecular weight of this protein is 26.3 kDa, which matched the result of SDS-PAGE.…”

Characterization and Low-Resolution Structure of an Extremely Thermostable Esterase of Potential Biotechnological Interest from Pyrococcus furiosus

“…Esterases often show broad substrate spectrum and are widely used as biocatalysts for the synthesis of important materials in pharmaceutical and chemical industries [33].…”