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Here, we combine deep learning\u2010based structure prediction with molecular dynamics simulations to guide loop\u2010centric engineering of a TdT from\n                    <jats:italic>Crocodylus porosus<\/jats:italic>\n                    . The resulting variant M3 (V253E\/L256M\/N338R) showed a 26\u2010fold increase in specific activity toward 3'\u2010ONH\n                    <jats:sub>2<\/jats:sub>\n                    \u2010dCTP while maintaining expression and thermostability. Simulations indicated that the mutations reorganized Loop1 dynamics and reproducibly biased the incoming nucleotide toward a more favorable in\u2010line attack orientation. The key N338R change also improved a second TdT scaffold, supporting N338 as a potentially transferable engineering hotspot. These results provide a practical computational strategy for improving terminal deoxynucleotidyl transferases for incorporation of 3\u2032\u2010modified nucleotides, reducing reliance on large\u2010scale screening.\n                  <\/jats:p>","DOI":"10.1002\/bit.70397","type":"journal-article","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T09:45:41Z","timestamp":1790761541000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Loop\u2010Centric Engineering of Terminal Deoxynucleotidyl Transferase for Enhanced Catalysis of 3'\u2010Modified Nucleotides"],"prefix":"10.1002","author":[{"given":"Kun","family":"Shi","sequence":"first","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering, School of Biotechnology East China University of Science and Technology Shanghai P.R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-0838-1026","authenticated-orcid":false,"given":"An\u2010Na","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering, School of Biotechnology East China University of Science and Technology Shanghai P.R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4301-7630","authenticated-orcid":false,"given":"Jian\u2010He","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering, School of Biotechnology East China University of Science and Technology Shanghai P.R. 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