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Here, we identify the features underlying the catalytic efficiency of a highly proficient\n                  <jats:italic>de novo<\/jats:italic>\n                  enzyme, from the original computational design to an optimized catalyst obtained through two rounds of directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, binding of a transition state analog also led to widespread increases in backbone conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. Also, the initial design considered only the transition state but not substrate binding, leading to a dynamic Michaelis complex prior to optimization. These studies show the multiple features that need to be optimized to achieve high activity in a designed enzyme.\n                <\/jats:p>","DOI":"10.64898\/2026.05.29.728685","type":"posted-content","created":{"date-parts":[[2026,5,30]],"date-time":"2026-05-30T01:26:03Z","timestamp":1780104363000},"source":"Crossref","is-referenced-by-count":0,"title":["Interplay of stability and dynamics in the optimization of a highly proficient\n                  <i>de novo<\/i>\n                  enzyme"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8020-9255","authenticated-orcid":false,"given":"Sagar","family":"Bhattacharya","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Chemistry & Cardiovascular Research Institute, University of California, San Francisco, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gessica M.","family":"Adornato","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pittsburgh, PA 15260, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuda","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry & Cardiovascular Research Institute, University of California, San Francisco, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Duke University, Durham, NC 27708, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wael E. Y.","family":"Mouloud","sequence":"additional","affiliation":[{"name":"Research Group of Organic Chemistry, Vrije Universiteit Brussel (VUB), Brussels, 1050, Belgium"},{"name":"Jean Jeener NMR Centre, Vrije Universiteit Brussel (VUB), Brussels, 1050, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyunil","family":"Jo","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry & Cardiovascular Research Institute, University of California, San Francisco, CA 94158, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander N.","family":"Volkov","sequence":"additional","affiliation":[{"name":"Jean Jeener NMR Centre, Vrije Universiteit Brussel (VUB), Brussels, 1050, Belgium"},{"name":"VIB-VUB Centre for Structural Biology, Vlaams Instituut voor Biotechnologie (VIB), Brussels, 1050, Belgium"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivan V.","family":"Korendovych","sequence":"additional","affiliation":[{"name":"Department of Chemistry & Biochemistry, Baylor University, Waco, TX 76798, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA 93106, USA"},{"name":"Department of Bioengineering, University of California Santa Barbara, Santa Barbara, CA 93106, USA"},{"name":"Howard Hughes Medical Institute, University of California Santa Barbara, Santa Barbara, CA 93106, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David N.","family":"Beratan","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Duke University, Durham, NC 27708, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Pittsburgh, PA 15260, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William F.","family":"DeGrado","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry & Cardiovascular Research Institute, University of California, San Francisco, CA 94158, USA"},{"name":"Department of Molecular and Chemical Sciences, Tohoku University, Sendai 980-8577, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091612051012000_2026.05.29.728685v3.1","doi-asserted-by":"publisher","DOI":"10.1038\/350121a0"},{"key":"2026091612051012000_2026.05.29.728685v3.2","doi-asserted-by":"publisher","DOI":"10.1021\/cr0503106"},{"key":"2026091612051012000_2026.05.29.728685v3.3","unstructured":"Fersht, A. 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