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Previously, the T23V\/Q213A mutant of AKR from\n                    <jats:italic>Kluyveromyces marxianus<\/jats:italic>\n                    (\n                    <jats:italic>Km<\/jats:italic>\n                    AKR) exhibited an extended substrate scope, but it still showed poor catalytic activity toward some valuable aliphatic and aromatic ketones. Here, we developed a computer\u2010assisted strategy to virtually screen a mutation library constructed from residues 23 and 213. Guided by the binding free energy calculated from high\u2010throughput molecular dynamics simulations, the top ten mutants with the lowest binding energies in each group were selected for testing against the corresponding substrates. It was found that most selected mutants exhibited enhanced catalytic activity, yielding the corresponding pharmaceutically important alcohols with high enantioselectivities. Structural and dynamic analyses indicated that residues 23 and 213 functioned as molecular switches to control the dynamics of the loop regions that constitute the substrate\u2010binding pocket, thereby influencing the substrate specificity of\n                    <jats:italic>Km<\/jats:italic>\n                    AKR.\n                  <\/jats:p>","DOI":"10.1002\/biot.70130","type":"journal-article","created":{"date-parts":[[2025,10,3]],"date-time":"2025-10-03T13:47:57Z","timestamp":1759499277000},"source":"Crossref","is-referenced-by-count":0,"title":["Molecular Dynamics Simulation\u2010Assisted Tuning of Key Sites to Alter the Substrate Specificity of the Aldo\u2010Keto Reductase\n                    <i>Km<\/i>\n                    AKR"],"prefix":"10.1002","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9903-1944","authenticated-orcid":false,"given":"Shen\u2010Yuan","family":"Xu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology  Hangzhou China"},{"name":"Zhejiang Key Laboratory of Bioorganic Synthesis College of Biotechnology and Bioengineering Zhejiang University of Technology  Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education Zhejiang University of Technology  Hangzhou China"},{"name":"The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals Zhejiang University of Technology  Hangzhou China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu\u2010Feng","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology  Hangzhou China"},{"name":"Zhejiang Key Laboratory of Bioorganic Synthesis College of Biotechnology and Bioengineering Zhejiang University of Technology  Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education Zhejiang University of Technology  Hangzhou China"},{"name":"The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals Zhejiang University of Technology  Hangzhou China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lei","family":"Cui","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology  Hangzhou China"},{"name":"Zhejiang Key Laboratory of Bioorganic Synthesis College of Biotechnology and Bioengineering Zhejiang University of Technology  Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education Zhejiang University of Technology  Hangzhou China"},{"name":"The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals Zhejiang University of Technology  Hangzhou 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