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Title: Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

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. Semi-rational 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. Moreover, 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. Based on thesemore » computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, 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 turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, 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.« less
 [1] ;  [1] ;  [2] ;  [1]
  1. East China Univ. of Science and Technology, Shanghai (China)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Publication Date:
OSTI Identifier:
Grant/Contract Number:
AC05-00OR22725; 2014M551349; 2011CB710800; 2011AA02A210; 2060204
Accepted Manuscript
Journal Name:
Additional Journal Information:
Journal Volume: 54; Journal Issue: 9; Journal ID: ISSN 0006-2960
American Chemical Society (ACS)
Research Org:
Oak Ridge National Laboratory (ORNL); Oak Ridge Leadership Computing Facility (OLCF)
Sponsoring Org:
Country of Publication:
United States
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY carboxylesterase; structural mechanism; molecular dynamics simulation; rational design