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. 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 »
- Authors:
-
- East China Univ. of Science and Technology, Shanghai (China)
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
- Publication Date:
- Research Org.:
- Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility (OLCF)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1185936
- Grant/Contract Number:
- AC05-00OR22725; 2014M551349; 2011CB710800; 2011AA02A210; 2060204
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biochemistry
- Additional Journal Information:
- Journal Volume: 54; Journal Issue: 9; Journal ID: ISSN 0006-2960
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; carboxylesterase; structural mechanism; molecular dynamics simulation; rational design
Citation Formats
Chen, Qi, Luan, Zheng-Jiao, Cheng, Xiaolin, and Xu, Jian-He. Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase. United States: N. p., 2015.
Web. doi:10.1021/bi5015612.
Chen, Qi, Luan, Zheng-Jiao, Cheng, Xiaolin, & Xu, Jian-He. Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase. United States. https://doi.org/10.1021/bi5015612
Chen, Qi, Luan, Zheng-Jiao, Cheng, Xiaolin, and Xu, Jian-He. Wed .
"Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase". United States. https://doi.org/10.1021/bi5015612. https://www.osti.gov/servlets/purl/1185936.
@article{osti_1185936,
title = {Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase},
author = {Chen, Qi and Luan, Zheng-Jiao and Cheng, Xiaolin and Xu, Jian-He},
abstractNote = {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 these 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.},
doi = {10.1021/bi5015612},
journal = {Biochemistry},
number = 9,
volume = 54,
place = {United States},
year = {Wed Feb 25 00:00:00 EST 2015},
month = {Wed Feb 25 00:00:00 EST 2015}
}
Web of Science
Works referenced in this record:
Structure, function and regulation of carboxylesterases
journal, September 2006
- Satoh, Tetsuo; Hosokawa, Masakiyo
- Chemico-Biological Interactions, Vol. 162, Issue 3
Microbial carboxyl esterases: classification, properties and application in biocatalysis
journal, March 2002
- Bornscheuer, Uwe T.
- FEMS Microbiology Reviews, Vol. 26, Issue 1
A thermostable exo-β-fructosidase immobilised through rational design
journal, February 2014
- Martínez, Duniesky; Cutiño-Avila, Bessy; Pérez, Enrique Rosendo
- Food Chemistry, Vol. 145
Reversal of coenzyme specificity and improvement of catalytic efficiency of Pichia stipitis xylose reductase by rational site-directed mutagenesis
journal, March 2009
- Zeng, Qi-Kai; Du, Hong-Li; Wang, Jing-Fang
- Biotechnology Letters, Vol. 31, Issue 7
Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis
journal, February 2013
- Irani, Mehdi; Törnvall, Ulrika; Genheden, Samuel
- Biochemistry, Vol. 52, Issue 7
Key Amino Acid Associated with Acephate Detoxification by Cydia pomonella Carboxylesterase Based on Molecular Dynamics with Alanine Scanning and Site-Directed Mutagenesis
journal, May 2014
- Yang, Xue Qing; Liu, Ji Yuan; Li, Xian Chun
- Journal of Chemical Information and Modeling, Vol. 54, Issue 5
Structure and stability of a thermostable carboxylesterase from the thermoacidophilic archaeon Sulfolobus tokodaii: Structural study on S. tokodaii carboxylesterase
journal, July 2012
- Angkawidjaja, Clement; Koga, Yuichi; Takano, Kazufumi
- FEBS Journal, Vol. 279, Issue 17
A thermostable and organic-solvent tolerant esterase from Pseudomonas putida ECU1011: Catalytic properties and performance in kinetic resolution of α-hydroxy acids
journal, April 2013
- Ma, Bao-Di; Yu, Hui-Lei; Pan, Jiang
- Bioresource Technology, Vol. 133
Antiplatelet therapy: new pharmacological agents and changing paradigms
journal, June 2013
- Capodanno, D.; Ferreiro, J. L.; Angiolillo, D. J.
- Journal of Thrombosis and Haemostasis, Vol. 11
Increased Catalyst Productivity in α-Hydroxy Acids Resolution by Esterase Mutation and Substrate Modification
journal, January 2014
- Ma, Bao-Di; Kong, Xu-Dong; Yu, Hui-Lei
- ACS Catalysis, Vol. 4, Issue 3
Crystal structures of Pseudomonas putida esterase reveal the functional role of residues 187 and 287 in substrate binding and chiral recognition
journal, April 2014
- Dou, Shuai; Kong, Xu-Dong; Ma, Bao-Di
- Biochemical and Biophysical Research Communications, Vol. 446, Issue 4
Alpha Beta-Hydrolase Fold Enzymes Structures, Functions and Mechanisms
journal, September 2000
- Holmquist, M.
- Current Protein and Peptide Science, Vol. 1, Issue 2
Evaluation of the ‘side door’ in carboxylesterase-mediated catalysis and inhibition
journal, February 2008
- Streit, Timothy M.; Borazjani, Abdolsamad; Lentz, Shellaine E.
- Biological Chemistry, Vol. 389, Issue 2
Molecular Modeling of CPT-11 Metabolism by Carboxylesterases (CEs): Use of pnb CE as a Model
journal, January 2004
- Wierdl, Monika; Morton, Christopher L.; Nguyen, Nathan K.
- Biochemistry, Vol. 43, Issue 7
Global and local molecular dynamics of a bacterial carboxylesterase provide insight into its catalytic mechanism
journal, November 2011
- Yu, Xiaozhen; Sigler, Sara C.; Hossain, Delwar
- Journal of Molecular Modeling, Vol. 18, Issue 6
Comparison of simple potential functions for simulating liquid water
journal, July 1983
- Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.
- The Journal of Chemical Physics, Vol. 79, Issue 2
Comparison of multiple Amber force fields and development of improved protein backbone parameters
journal, November 2006
- Hornak, Viktor; Abel, Robert; Okur, Asim
- Proteins: Structure, Function, and Bioinformatics, Vol. 65, Issue 3
The Amber biomolecular simulation programs
journal, January 2005
- Case, David A.; Cheatham, Thomas E.; Darden, Tom
- Journal of Computational Chemistry, Vol. 26, Issue 16
Scalable molecular dynamics with NAMD
journal, January 2005
- Phillips, James C.; Braun, Rosemary; Wang, Wei
- Journal of Computational Chemistry, Vol. 26, Issue 16, p. 1781-1802
Particle mesh Ewald: An N ⋅log( N ) method for Ewald sums in large systems
journal, June 1993
- Darden, Tom; York, Darrin; Pedersen, Lee
- The Journal of Chemical Physics, Vol. 98, Issue 12
GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit
journal, February 2013
- Pronk, Sander; Páll, Szilárd; Schulz, Roland
- Bioinformatics, Vol. 29, Issue 7
Bio3d: an R package for the comparative analysis of protein structures
journal, August 2006
- Grant, B. J.; Rodrigues, A. P. C.; ElSawy, K. M.
- Bioinformatics, Vol. 22, Issue 21
Calculating Structures and Free Energies of Complex Molecules: Combining Molecular Mechanics and Continuum Models
journal, December 2000
- Kollman, Peter A.; Massova, Irina; Reyes, Carolina
- Accounts of Chemical Research, Vol. 33, Issue 12
Impact of Resistance Mutations on Inhibitor Binding to HIV-1 Integrase
journal, November 2013
- Chen, Qi; Buolamwini, John K.; Smith, Jeremy C.
- Journal of Chemical Information and Modeling, Vol. 53, Issue 12
LigPlot+: Multiple Ligand–Protein Interaction Diagrams for Drug Discovery
journal, October 2011
- Laskowski, Roman A.; Swindells, Mark B.
- Journal of Chemical Information and Modeling, Vol. 51, Issue 10
Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins: Sequence conservation and 3 0 structure
journal, March 1993
- Cygler, Miroslaw; Schrag, Joseph D.; Sussman, Joel L.
- Protein Science, Vol. 2, Issue 3
A low-barrier hydrogen bond in the catalytic triad of serine proteases
journal, June 1994
- Frey, P.; Whitt, S.; Tobin, J.
- Science, Vol. 264, Issue 5167
Ground State and Transition State Contributions to the Rates of Intramolecular and Enzymatic Reactions
journal, February 1999
- Bruice, Thomas C.; Lightstone, Felice C.
- Accounts of Chemical Research, Vol. 32, Issue 2
Role of the N Terminus in Enzyme Activity, Stability and Specificity in Thermophilic Esterases Belonging to the HSL Family
journal, January 2005
- Mandrich, Luigi; Merone, Luigia; Pezzullo, Margherita
- Journal of Molecular Biology, Vol. 345, Issue 3
High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement
journal, March 2008
- Park, Suk-Youl; Lee, Sang-Hak; Lee, Jieun
- Journal of Molecular Biology, Vol. 376, Issue 5
Clustal W and Clustal X version 2.0
journal, September 2007
- Larkin, M. A.; Blackshields, G.; Brown, N. P.
- Bioinformatics, Vol. 23, Issue 21, p. 2947-2948
SuSPect: Enhanced Prediction of Single Amino Acid Variant (SAV) Phenotype Using Network Features
journal, July 2014
- Yates, Christopher M.; Filippis, Ioannis; Kelley, Lawrence A.
- Journal of Molecular Biology, Vol. 426, Issue 14
Works referencing / citing this record:
Identification and characterization of a novel carboxylesterase from Phaseolus vulgaris for detection of organophosphate and carbamates pesticides: Identification and characterization of a novel carboxylesterase from P. vulgaris
journal, May 2018
- Yang, Xiao; Dai, Juan; Zhao, Sujuan
- Journal of the Science of Food and Agriculture, Vol. 98, Issue 13
A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys
journal, March 2018
- Gao, Haofeng; Li, Chanjuan; Bandikari, Ramesh
- Microbial Cell Factories, Vol. 17, Issue 1
Functional analysis of four upregulated carboxylesterase genes associated with fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval): Esterase genes lead to fenpropathrin resistance in Tetranychus cinnabarinus
journal, August 2018
- Wei, Peng; Li, Jinhang; Liu, Xinyang
- Pest Management Science, Vol. 75, Issue 1
Downregulation of carboxylesterase contributes to cyflumetofen resistance in Tetranychus cinnabarinus (Boisduval): Downregulated esterase genes in cyflumetofen resistance in mites
journal, March 2019
- Wei, Peng; Chen, Ming; Nan, Can
- Pest Management Science, Vol. 75, Issue 8
Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site
journal, February 2016
- Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon
- Extremophiles, Vol. 20, Issue 2
Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei
journal, June 2015
- Yang, Shaoqing; Qin, Zhen; Duan, Xiaojie
- Journal of Lipid Research, Vol. 56, Issue 8
Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1
journal, December 2017
- Kashif, Ayesha; Tran, Lan-Huong; Jang, Sei-Heon
- ACS Omega, Vol. 2, Issue 12
A novel cold-adapted esterase from Enterobacter cloacae: Characterization and improvement of its activity and thermostability via the site of Tyr193Cys
journal, March 2018
- Gao, Haofeng; Li, Chanjuan; Bandikari, Ramesh
- Microbial Cell Factories, Vol. 17, Issue 1
Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei
journal, June 2015
- Yang, Shaoqing; Qin, Zhen; Duan, Xiaojie
- Journal of Lipid Research, Vol. 56, Issue 8
Gut-brain axis metabolic pathway regulates antidepressant efficacy of albiflorin
journal, January 2018
- Zhao, Zhen-Xiong; Fu, Jie; Ma, Shu-Rong
- Theranostics, Vol. 8, Issue 21