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In this study, we demonstrate bidirectional enantioselectivity switch of HHDHs for the asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3\u2010dichloro\u20102\u2010propanol (1,3\u2010DCP). HHDH from\n                    <jats:italic>Propionicicella superfundia<\/jats:italic>\n                    (\n                    <jats:italic>Ps<\/jats:italic>\n                    HHDH) was identified for its high activity toward 1,3\u2010DCP with a slight preference for (\n                    <jats:italic>R<\/jats:italic>\n                    )\u2010ECH formation (13.6%\n                    <jats:italic>ee<\/jats:italic>\n                    ). Guided by the catalytic mechanism of HHDHs and substrate binding features, we designed a rational engineering strategy targeting two distinct cavities within the active pocket of\n                    <jats:italic>Ps<\/jats:italic>\n                    HHDH to achieve controllable enantioselectivity. Through site\u2010saturation and iterative mutagenesis, we obtained two complementary variants: N87K\/R88F, producing (\n                    <jats:italic>R<\/jats:italic>\n                    )\u2010ECH with 96.2%\n                    <jats:italic>ee<\/jats:italic>\n                    , and N178Y\/Y242F\/L243Y, producing (\n                    <jats:italic>S<\/jats:italic>\n                    )\u2010ECH with 80.7%\n                    <jats:italic>ee<\/jats:italic>\n                    . Molecular dynamics simulations and quantum mechanics analyses revealed that residue substitutions in these cavities reshape the S\n                    <jats:sub>N<\/jats:sub>\n                    2 reaction trajectory, modulate halide ion release channels, and optimize active pocket volume, and thus collectively determining enantioselectivity. This strategy was successfully applied to four additional HHDHs, demonstrating its broad applicability. Notably, the N87K\/R88F variant produced 90\u2009mM (\n                    <jats:italic>R<\/jats:italic>\n                    )\u2010ECH within 80\u2009min using whole cell catalysts, highlighting its potential for industrial applications. Our work presents a structure\u2010guided framework for precise control of enantioselectivity in HHDHs and provides mechanistic insight into stereoselective catalysis.\n                  <\/jats:p>","DOI":"10.1002\/bit.70151","type":"journal-article","created":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T06:30:31Z","timestamp":1767594631000},"page":"983-994","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Controlling Enantioselectivity of Halohydrin Dehalogenase for Asymmetric Synthesis of Chiral Epichlorohydrin"],"prefix":"10.1002","volume":"123","author":[{"given":"Sheng","family":"Cai","sequence":"first","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology Hangzhou Zhejiang P. R. China"},{"name":"Zhejiang Key Laboratory of Bioorganic Synthesis, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang P. R. China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of Ministry of Education Zhejiang University of Technology Hangzhou Zhejiang P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian\u2010Qiang","family":"Jin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology Hangzhou Zhejiang P. R. China"},{"name":"Zhejiang Key Laboratory of Bioorganic Synthesis, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang P. R. China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of Ministry of Education Zhejiang University of Technology Hangzhou Zhejiang P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7485-5544","authenticated-orcid":false,"given":"Xiao\u2010Ling","family":"Tang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology Hangzhou Zhejiang P. R. China"},{"name":"Zhejiang Key Laboratory of Bioorganic Synthesis, College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang P. R. China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of Ministry of Education Zhejiang University of Technology Hangzhou Zhejiang P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ren\u2010Chao","family":"Zheng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Zhejiang University of Technology Hangzhou Zhejiang P. R. 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