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Nevertheless, protein delivery of yCD is challenging in part due to its thermal instability. Herein, we have rationally engineered a mutant yCD by replacing Met100 situated at the active site entry with the bulkier histidine to hinder potential oxidation of the active site Cys91. The engineered yCD-Met100His exhibits significantly enhanced activity and thermal stability. yCD-M100H is then genetically fused to the crystal-forming protein Cry3Aa to generate Cry3Aa-yCD-M100H fusion crystals to facilitate the enzyme\u2019s uptake into macrophages. The resulting Cry3Aa-yCD-M100H-loaded macrophages exhibit excellent penetration into tumor spheroids and readily convert 5-FC to 5-FU leading to efficacious cancer cell killing. This study showcases a promising route for stabilizing yCD and the feasibility of enzyme-internalized macrophages to serve as tumor-specific enzyme\/prodrug activators.<\/jats:p>","DOI":"10.1038\/s42003-025-08931-x","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T18:38:54Z","timestamp":1763059134000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["An engineered yeast cytosine deaminase with improved catalytic activity and stability for macrophage-mediated enzyme\/prodrug therapy"],"prefix":"10.1038","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1637-3090","authenticated-orcid":false,"given":"Jiale","family":"Zheng","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahao","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristen Wing Yu","family":"Yung","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2220-3116","authenticated-orcid":false,"given":"Qipeng","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8627-1236","authenticated-orcid":false,"given":"Marianne M.","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9758-3243","authenticated-orcid":false,"given":"Michael K.","family":"Chan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,13]]},"reference":[{"key":"8931_CR1","doi-asserted-by":"publisher","first-page":"330","DOI":"10.1038\/nrc1074","volume":"3","author":"DB Longley","year":"2003","unstructured":"Longley, D. 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