{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T12:49:37Z","timestamp":1791809377542,"version":"4.3.4"},"reference-count":79,"publisher":"American Chemical Society (ACS)","issue":"19","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002367","name":"Chinese Academy of Sciences","doi-asserted-by":"publisher","award":["306GJHZ2025003BS"],"award-info":[{"award-number":["306GJHZ2025003BS"]}],"id":[{"id":"10.13039\/501100002367","id-type":"DOI","asserted-by":"publisher"}]},{"name":"H2020 Marie Sklodowska-Curie Actions","award":["722610"],"award-info":[{"award-number":["722610"]}]},{"name":"National Basic Research Program of China (973 Program)","award":["2021YFA0911500"],"award-info":[{"award-number":["2021YFA0911500"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Molecular dynamics (MD)-based methods to predict the enantioselectivity or product specificity of enzymes typically use a near attack conformation (NAC) approach. The computational efficiency of this ground-state approach, taking minutes to hours, allows to in silico screen thousands of enzyme variants. During the computational design and screening of new variants of limonene epoxide hydrolase for improved stereoselectivity, it was noticed that, while this approach gave satisfactory prediction accuracy for most substrates, the enantioselectivity predictions were far less accurate for variants designed for cis-stilbene oxide. Here, to investigate whether inaccurate predictions with the NAC-based approach were typical for this substrate, first, a larger data set was generated by experimentally testing and computationally predicting the enantioselectivity of an additional 18 enzyme variants for cis-stilbene oxide. Subsequent MD simulations confirmed that a NAC analysis resulted in inaccurate enantioselectivity predictions throughout the data set. Density functional theory (DFT) calculations using the B3LYP-D3(BJ) functional, with an active site cluster model of &amp;gt;300 atoms, provided a rationale for why the NAC approach gave poor predictions with cis-stilbene oxide. The aromatic rings of the substrate influence the nucleophilic attack on the epoxide group, by providing a blend of stereoelectronic activation and steric hindrance to the transition state, which is not considered in the NAC approach. With this substrate, the DFT accurately predicted the enantioselectivity. The results offer valuable insights into the applicability of the NAC approach for predicting enzyme selectivity, suggesting that more expensive methodologies, which do explicitly consider the transition state structure, can also become required when bulky or stereoelectronically activating groups are bound to the reacting atoms.<\/jats:p>","DOI":"10.1021\/acs.jcim.6c02029","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:01:44Z","timestamp":1790092904000},"page":"12877-12890","source":"Crossref","is-referenced-by-count":0,"title":["Unraveling a Case Where the NAC Approach Fails\u2500MD\nand QM Modeling of Regioselective\n                    <i>cis<\/i>\n                    -Stilbene Oxide\nHydrolysis by Engineered Epoxide Hydrolases"],"prefix":"10.1021","volume":"66","author":[{"given":"Elvira","family":"Bombino","sequence":"first","affiliation":[{"name":"University of Groningen , , Nijenborgh 4 , ,","place":["Groningen, The Netherlands, 9747AG"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hesam","family":"Arabnejad","sequence":"additional","affiliation":[{"name":"University of Groningen , , Nijenborgh 4 , ,","place":["Groningen, The Netherlands, 9747AG"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baoyan","family":"Liu","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, P. R. China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter A.","family":"Jekel","sequence":"additional","affiliation":[{"name":"University of Groningen , , Nijenborgh 4 , ,","place":["Groningen, The Netherlands, 9747AG"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6542-6649","authenticated-orcid":true,"given":"Xiang","family":"Sheng","sequence":"additional","affiliation":[{"name":"Chinese Academy of Sciences , , ,","place":["Tianjin, P. R. China, 300308"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0834-2043","authenticated-orcid":true,"given":"Dick B.","family":"Janssen","sequence":"additional","affiliation":[{"name":"University of Groningen , , Nijenborgh 4 , ,","place":["Groningen, The Netherlands, 9747AG"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1012-5611","authenticated-orcid":true,"given":"Fahmi","family":"Himo","sequence":"additional","affiliation":[{"name":"Stockholm University , , ,","place":["Stockholm, Sweden, 10691"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0891-6972","authenticated-orcid":true,"given":"Hein J.","family":"Wijma","sequence":"additional","affiliation":[{"name":"University of Groningen , , Nijenborgh 4 , ,","place":["Groningen, The Netherlands, 9747AG"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"issue":"7397","key":"2026101208073022400_cit1","doi-asserted-by":"publisher","first-page":"185","DOI":"10.1038\/nature11117","article-title":"Engineering the Third Wave of Biocatalysis","volume":"485","author":"Bornscheuer","year":"2012","journal-title":"Nature"},{"issue":"2","key":"2026101208073022400_cit2","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1016\/j.cbpa.2010.11.011","article-title":"Status of Protein Engineering for\nBiocatalysts: How to Design an Industrially Useful Biocatalyst","volume":"15","author":"Bommarius","year":"2011","journal-title":"Curr. Opin. Chem. Biol."},{"issue":"2110","key":"2026101208073022400_cit3","doi-asserted-by":"publisher","first-page":"20170062","DOI":"10.1098\/rsta.2017.0062","article-title":"Integrating\nProtein Engineering with Process Design for Biocatalysis","volume":"376","author":"Woodley","year":"2018","journal-title":"Philos. Trans A Math Phys. Eng. Sci."},{"issue":"10","key":"2026101208073022400_cit4","doi-asserted-by":"publisher","first-page":"1117","DOI":"10.3390\/catal10101117","article-title":"Epoxide Syntheses\nand Ring-Opening Reactions in Drug Development","volume":"10","author":"Moschona","year":"2020","journal-title":"Catalysts"},{"key":"2026101208073022400_cit5","doi-asserted-by":"publisher","first-page":"105862","DOI":"10.1016\/j.bioorg.2022.105862","article-title":"Epoxides Developability\nas Active Pharmaceutical Ingredients and\nBiochemical Probes","volume":"125","author":"Kaur","year":"2022","journal-title":"Bioorg. Chem."},{"issue":"6","key":"2026101208073022400_cit6","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1021\/ar960061v","article-title":"Asymmetric\nCatalysis of Epoxide Ring-Opening Reactions","volume":"33","author":"Jacobsen","year":"2000","journal-title":"Acc. Chem. Res."},{"issue":"4","key":"2026101208073022400_cit7","doi-asserted-by":"publisher","first-page":"414","DOI":"10.1016\/S0958-1669(03)00102-2","article-title":"Biocatalytic Conversion of Epoxides","volume":"14","author":"de Vries","year":"2003","journal-title":"Curr. Opin. Biotechnol."},{"issue":"4","key":"2026101208073022400_cit8","doi-asserted-by":"publisher","first-page":"2905","DOI":"10.1128\/AEM.72.4.2905-2917.2006","article-title":"Diversity\nand Biocatalytic Potential of Epoxide Hydrolases Identified by Genome\nAnalysis","volume":"72","author":"van Loo","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"2026101208073022400_cit9","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1007\/3-540-69791-8_7","article-title":"Epoxide Hydrolases\nand Their Synthetic Applications","volume":"63","author":"Orru","year":"1999","journal-title":"Adv. Biochem\nEng. Biotechnol"},{"issue":"4","key":"2026101208073022400_cit10","doi-asserted-by":"publisher","first-page":"365","DOI":"10.1081\/DMR-120026498","article-title":"The Telltale Structures of Epoxide Hydrolases","volume":"35","author":"Arand","year":"2003","journal-title":"Drug Metab. Rev."},{"issue":"19","key":"2026101208073022400_cit11","doi-asserted-by":"publisher","first-page":"5052","DOI":"10.1128\/JB.180.19.5052-5057.1998","article-title":"Limonene-1,2-Epoxide\nHydrolase from Rhodococcus Erythropolis DCL14 Belongs to a Novel Class\nof Epoxide Hydrolases","volume":"180","author":"van der Werf","year":"1998","journal-title":"J. Bacteriol."},{"issue":"11","key":"2026101208073022400_cit12","doi-asserted-by":"publisher","first-page":"2583","DOI":"10.1093\/emboj\/cdg275","article-title":"Structure\nof Rhodococcus Erythropolis Limonene-1,2-Epoxide Hydrolase Reveals\na Novel Active Site","volume":"22","author":"Arand","year":"2003","journal-title":"EMBO J."},{"issue":"41","key":"2026101208073022400_cit13","doi-asserted-by":"publisher","first-page":"14339","DOI":"10.1021\/ja050940p","article-title":"Catalytic Mechanism\nof Limonene Epoxide\nHydrolase, a Theoretical Study","volume":"127","author":"Hopmann","year":"2005","journal-title":"J. Am. Chem.\nSoc."},{"issue":"17","key":"2026101208073022400_cit14","doi-asserted-by":"publisher","first-page":"4563","DOI":"10.1002\/anie.201300594","article-title":"Quantum Chemistry\nas a Tool in Asymmetric Biocatalysis:\nLimonene Epoxide Hydrolase Test Case","volume":"52","author":"Lind","year":"2013","journal-title":"Angew.\nChem. Int. Ed."},{"issue":"15","key":"2026101208073022400_cit15","doi-asserted-by":"publisher","first-page":"2879","DOI":"10.1111\/febs.13328","article-title":"Discovery and Characterization\nof Thermophilic Limonene-1,2-Epoxide Hydrolases from Hot Spring Metagenomic\nLibraries","volume":"282","author":"Ferrandi","year":"2015","journal-title":"FEBS J."},{"issue":"1","key":"2026101208073022400_cit16","doi-asserted-by":"publisher","first-page":"138","DOI":"10.1002\/anie.201000826","article-title":"Laboratory\nEvolution of Stereoselective Enzymes: A Prolific Source of Catalysts\nfor Asymmetric Reactions","volume":"50","author":"Reetz","year":"2011","journal-title":"Angew. Chem., Int.\nEd."},{"issue":"44","key":"2026101208073022400_cit17","doi-asserted-by":"publisher","first-page":"15744","DOI":"10.1021\/ja1067542","article-title":"Manipulating the\nStereoselectivity of Limonene Epoxide\nHydrolase by Directed Evolution Based on Iterative Saturation Mutagenesis","volume":"132","author":"Zheng","year":"2010","journal-title":"J. Am. Chem. 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Enzymol."},{"issue":"6","key":"2026101208073022400_cit24","doi-asserted-by":"publisher","first-page":"3031","DOI":"10.1021\/acs.jctc.7b00125","article-title":"The Rosetta\nAll-Atom Energy Function for Macromolecular Modeling and Design","volume":"13","author":"Alford","year":"2017","journal-title":"J. Chem. Theory Comput."},{"issue":"5","key":"2026101208073022400_cit25","doi-asserted-by":"publisher","first-page":"2915","DOI":"10.1021\/acscatal.9b05223","article-title":"Robust \u03c9-Transaminases by Computational\nStabilization of the Subunit Interface","volume":"10","author":"Meng","year":"2020","journal-title":"ACS\nCatal."},{"key":"2026101208073022400_cit26","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.54639","article-title":"Approaching Boiling Point Stability\nof an Alcohol Dehydrogenase through Computationally-Guided Enzyme\nEngineering","volume":"9","author":"Aalbers","year":"2020","journal-title":"eLife"},{"issue":"3","key":"2026101208073022400_cit27","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1093\/protein\/gzw068","article-title":"A Robust\nCosolvent-Compatible Halohydrin Dehalogenase by Computational Library\nDesign","volume":"30","author":"Arabnejad","year":"2016","journal-title":"Protein Eng., Des. 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J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J.\n          Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford CT, 2016."},{"issue":"7","key":"2026101208073022400_cit37","doi-asserted-by":"publisher","first-page":"2079","DOI":"10.1021\/ci500126x","article-title":"Computationally\nEfficient and Accurate\nEnantioselectivity Modeling by Clusters of Molecular Dynamics Simulations","volume":"54","author":"Wijma","year":"2014","journal-title":"J. Chem. Inf. 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Eur. J."},{"issue":"20","key":"2026101208073022400_cit41","doi-asserted-by":"publisher","first-page":"5016","DOI":"10.1111\/febs.16906","article-title":"Regio- and Stereoselective Steroid Hydroxylation by\nCYP109A2 from Bacillus Megaterium Explored by X-ray\nCrystallography and Computational Modeling","volume":"290","author":"Jo\u0301z\u0301wik","year":"2023","journal-title":"FEBS J."},{"issue":"15","key":"2026101208073022400_cit42","doi-asserted-by":"publisher","first-page":"154104","DOI":"10.1063\/1.3382344","article-title":"A Consistent and Accurate Ab Initio Parametrization\nof Density Functional Dispersion\nCorrection (DFT-D) for the 94 Elements H-Pu","volume":"132","author":"Grimme","year":"2010","journal-title":"J. Chem. Phys."},{"issue":"7","key":"2026101208073022400_cit43","doi-asserted-by":"publisher","first-page":"5648","DOI":"10.1063\/1.464913","article-title":"Density-Functional\nThermochemistry. III. The Role of Exact Exchange","volume":"98","author":"Becke","year":"1993","journal-title":"J. Chem. Phys."},{"issue":"2","key":"2026101208073022400_cit44","doi-asserted-by":"publisher","first-page":"785","DOI":"10.1103\/PhysRevB.37.785","article-title":"Development of the Colle-Salvetti\nCorrelation-Energy Formula into a Functional of the Electron Density","volume":"37","author":"Lee","year":"1988","journal-title":"Phys. Rev. B"},{"issue":"15","key":"2026101208073022400_cit45","doi-asserted-by":"publisher","first-page":"1787","DOI":"10.1002\/jcc.20495","article-title":"Semiempirical\nGGA-Type Density Functional Constructed with a Long-Range Dispersion\nCorrection","volume":"27","author":"Grimme","year":"2006","journal-title":"J. Comput. 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