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While the active site architecture and substrate specificity of\n                  <jats:italic>Av<\/jats:italic>\n                  PAL have been extensively characterized, the dynamic behavior of this unstructured loop beyond its role in catalysis remains poorly understood. Here, we investigate the functional role of this loop by restricting its mobility through targeted interchain disulfide bond engineering. Three in-house approaches were designed to predict ideal cysteine residue pairs: (i) quantifying pair interaction energies via electrostatic and van der Waals forces, (ii) generating a contact map of residues within 5 \u00c5 proximity, and (iii) implementing a machine-learning model trained on datasets from PDBCYS, SPX, and an internal database to rank cysteine pair likelihood within disulfide bond geometric constraints. Our machine-learning-guided strategy yielded a successful variant with complete oxidation efficiency in\n                  <jats:italic>E. coli<\/jats:italic>\n                  . Rigidification of this loop reveals that it also functions as a regulator of substrate specificity. Multi-scale molecular simulation analyses (molecular dynamics, metadynamics, quantum\/molecular mechanics) reveal that this modification alters the active-site pocket by reducing the conformational dynamics of substrate binding. Our findings underscore the delicate balance between enzyme flexibility and catalytic efficiency, providing novel insights into the role of this understudied dynamic loop region in\n                  <jats:italic>Av<\/jats:italic>\n                  PAL.\n                <\/jats:p>","DOI":"10.64898\/2026.05.01.722275","type":"posted-content","created":{"date-parts":[[2026,5,6]],"date-time":"2026-05-06T21:20:28Z","timestamp":1778102428000},"source":"Crossref","is-referenced-by-count":0,"title":["An engineered disulfide staple restricts lid loop dynamics and alters substrate specificity of phenylalanine ammonia-lyase"],"prefix":"10.64898","author":[{"given":"Rebecca","family":"Condruti","sequence":"first","affiliation":[{"name":"Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Likith","family":"Muthuraj","sequence":"additional","affiliation":[{"name":"Department of Protein Design and AI, Kcat Enzymatic Private Limited, Bengaluru 560005, Karnataka, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeevan K.","family":"Prakash","sequence":"additional","affiliation":[{"name":"Department of Protein Design and AI, Kcat Enzymatic Private Limited, Bengaluru 560005, Karnataka, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samuel D.","family":"Littman","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Tufts University, Medford, MA 02155, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pravin","family":"Kumar R.","sequence":"additional","affiliation":[{"name":"Department of Protein Design and AI, Kcat Enzymatic Private Limited, Bengaluru 560005, Karnataka, India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7737-1385","authenticated-orcid":false,"given":"Nikhil U.","family":"Nair","sequence":"additional","affiliation":[{"name":"Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, United States"},{"name":"Department of Chemical & Biological Engineering, Tufts University, Medford, MA 02155, United States"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026050812001692000_2026.05.01.722275v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2008.05.025"},{"key":"2026050812001692000_2026.05.01.722275v1.2","doi-asserted-by":"publisher","DOI":"10.1021\/ja0762689"},{"key":"2026050812001692000_2026.05.01.722275v1.3","doi-asserted-by":"crossref","first-page":"288","DOI":"10.1007\/s11244-018-0898-1","article-title":"Discovery and Investigation of Mutase-like Activity in a Phenylalanine Ammonia Lyase from Anabaena variabilis","volume":"61","year":"2018","journal-title":"Top Catal"},{"key":"2026050812001692000_2026.05.01.722275v1.4","doi-asserted-by":"publisher","DOI":"10.1002\/cctc.201200871"},{"key":"2026050812001692000_2026.05.01.722275v1.5","doi-asserted-by":"publisher","DOI":"10.1039\/d0cc00783h"},{"key":"2026050812001692000_2026.05.01.722275v1.6","doi-asserted-by":"publisher","DOI":"10.1021\/acscatal.1c05508"},{"key":"2026050812001692000_2026.05.01.722275v1.7","doi-asserted-by":"crossref","first-page":"541","DOI":"10.3390\/antibiotics9090541","article-title":"Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds","volume":"9","year":"2020","journal-title":"Antibiotics (Basel"},{"key":"2026050812001692000_2026.05.01.722275v1.8","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-14-346"},{"key":"2026050812001692000_2026.05.01.722275v1.9","doi-asserted-by":"publisher","DOI":"10.1002\/pro.5560021002"},{"key":"2026050812001692000_2026.05.01.722275v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2023.123440"},{"key":"2026050812001692000_2026.05.01.722275v1.11","doi-asserted-by":"crossref","first-page":"2057","DOI":"10.1080\/10408398.2021.1970508","article-title":"Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges","volume":"63","year":"2023","journal-title":"Critical Reviews in Food Science and Nutrition"},{"key":"2026050812001692000_2026.05.01.722275v1.12","doi-asserted-by":"publisher","DOI":"10.1016\/J.BEJ.2018.06.003"},{"key":"2026050812001692000_2026.05.01.722275v1.13","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1021\/ar960078d","article-title":"Toward Understanding and Tailoring the Specificity of Synthetically Useful Enzymes","volume":"32","year":"1999","journal-title":"Acc. 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