{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:52:10Z","timestamp":1760057530099,"version":"build-2065373602"},"reference-count":76,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,2,11]],"date-time":"2025-02-11T00:00:00Z","timestamp":1739232000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["21571019","21573020","21503018","2019YFC1805600"],"award-info":[{"award-number":["21571019","21573020","21503018","2019YFC1805600"]}]},{"name":"the National Key Research and Development Program of China","award":["21571019","21573020","21503018","2019YFC1805600"],"award-info":[{"award-number":["21571019","21573020","21503018","2019YFC1805600"]}]},{"name":"the Fundamental Research Funds for the Central Universities","award":["21571019","21573020","21503018","2019YFC1805600"],"award-info":[{"award-number":["21571019","21573020","21503018","2019YFC1805600"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>CYP199A4 is a cytochrome P450 and can catalyze the hydroxylation of 4-propionylbenzoic acid (4-pIBA) to generate \u03b1-hydroxyketone with high stereoselectivity. The F182L mutant of CYP199A4 (F182L-CYP199A4) has been shown to support the cleavage of the C\u2013C bond between the carbonyl and hydroxyl groups of \u03b1-hydroxyketone, whereas wild-type CYP199A4 cannot. To uncover how the Phe182 regulates substrate reactivity, we conducted classical molecular dynamics (MD) and quantum mechanics\/molecular mechanics (QM\/MM) MD simulations on these systems. The results predicted that the formation of \u03b1-hydroxyketone preferentially led to the (S)-enantiomer. Moreover, the findings revealed that the F182L-CYP199A4 facilitated the formation of a hydrogen bond between the \u03b1-hydroxyketone and the reactive peroxoanion (POA) species. This interaction stabilized the \u03b1-hydroxyketone near POA and promoted the subsequent C\u2013C bond cleavage. The mechanism of \u03b1-hydroxyketone formation and the subsequent C\u2013C bond cleavage were elucidated by employing the hybrid density functional theory (DFT). The \u03b1-hydroxyketone formation mechanism involved C\u2013H hydroxylation of 4-pIBA with a rate-limiting energy barrier of 17.1 kcal\/mol. The C\u2013C bond cleavage of \u03b1-hydroxyketone catalyzed by F182L-CYP199A4 occurred via a radical attack mechanism.<\/jats:p>","DOI":"10.3390\/ijms26041526","type":"journal-article","created":{"date-parts":[[2025,2,11]],"date-time":"2025-02-11T11:54:06Z","timestamp":1739274846000},"page":"1526","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mechanistic Insights into CYP199A4-Catalyzed \u03b1-Hydroxyketone Formation and Hydrogen Bond-Assisted C\u2013C Bond Cleavage Catalyzed by the CYP199A4 F182L Mutant"],"prefix":"10.3390","volume":"26","author":[{"given":"Chang","family":"Yuan","sequence":"first","affiliation":[{"name":"Institute of New Materials & Industrial Technologies, Wenzhou University, Wenzhou 325024, China"},{"name":"Key Laboratories of Theoretical and Computational Photochemistry, Ministry of Education, Beijing Normal University, Beijing 100875, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaqi","family":"Xu","sequence":"additional","affiliation":[{"name":"Key Laboratories of Theoretical and Computational Photochemistry, Ministry of Education, Beijing Normal University, Beijing 100875, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shun","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of New Materials & Industrial Technologies, Wenzhou University, Wenzhou 325024, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ye-Guang","family":"Fang","sequence":"additional","affiliation":[{"name":"Institute of New Materials & Industrial Technologies, Wenzhou University, Wenzhou 325024, China"},{"name":"Key Laboratories of Theoretical and Computational Photochemistry, Ministry of Education, Beijing Normal University, Beijing 100875, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1376-6019","authenticated-orcid":false,"given":"Hongwei","family":"Tan","sequence":"additional","affiliation":[{"name":"Key Laboratories of Theoretical and Computational Photochemistry, Ministry of Education, Beijing Normal University, Beijing 100875, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"12313","DOI":"10.1021\/acs.chemrev.3c00219","article-title":"Carbon-Carbon Bond Cleavage for Late-Stage Functionalization","volume":"123","author":"Liang","year":"2023","journal-title":"Chem. 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