{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:00:07Z","timestamp":1785416407480,"version":"3.56.0"},"reference-count":86,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T00:00:00Z","timestamp":1780444800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100007129","name":"Shandong Provincial Natural Science Foundation","doi-asserted-by":"publisher","award":["ZR2025QC1379"],"award-info":[{"award-number":["ZR2025QC1379"]}],"id":[{"id":"10.13039\/501100007129","id-type":"DOI","asserted-by":"publisher"}]},{"award":["ZR2025QC1379"],"award-info":[{"award-number":["ZR2025QC1379"]}],"id":[{"id":"https:\/\/ror.org\/04aqat463","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Shandong Province Traditional Chinese Medicine Science &amp; Technology Project","award":["M20242211"],"award-info":[{"award-number":["M20242211"]}]},{"name":"Scientific Research Fund Project of Shandong University of Traditional Chinese Medicine","award":["KYZK2024Q07"],"award-info":[{"award-number":["KYZK2024Q07"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Carnitine plays an essential role in fatty acid metabolism, and its biosynthesis is tightly regulated by \u03b3-butyrobetaine hydroxylase (BBOX), an Fe(II)\/\u03b1-ketoglutarate-dependent dioxygenase. BBOX is the target of mildronate (THP), a clinically used drug for treating ischemic heart diseases. However, the detailed mechanisms of BBOX-catalyzed hydroxylation and the atypical oxidative rearrangement underlying THP inhibition remain elusive. In this study, we employed combined quantum mechanics\/molecular mechanics (QM\/MM) methods to systematically elucidate these mechanisms at the atomic level. Our calculations reveal that the hydroxylation of \u03b3BB proceeds via a classical three-step mechanism in the quintet state, with hydrogen atom abstraction as the rate-determining step. Remarkably, substitution of the C4 methylene group in \u03b3BB with an amino group in THP redirects the reaction pathway, as the lone pair electrons on the adjacent nitrogen atom render N-N bond cleavage kinetically favored over hydroxyl rebound, thereby blocking carnitine synthesis. Through systematic evaluation of possible rearrangement pathways, we rule out the previously proposed direct 1,2-H migration and suggest a revised mechanism featuring imine-mediated hydrogen transfer, hydroxyl rebound preceding C-C bond formation, and final radical coupling. This work provides a detailed atomic-level understanding of both the catalytic and inhibitory mechanisms of BBOX, revealing how substrate electronic effects dictate reaction outcomes. The elucidated mechanistic insights offer a theoretical foundation for understanding the catalytic versatility of the \u03b1KG-dependent dioxygenase family and provide valuable guidance for the rational design of novel BBOX inhibitors.<\/jats:p>","DOI":"10.3390\/molecules31111941","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T13:46:48Z","timestamp":1780494408000},"page":"1941","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mechanistic Elucidation of BBOX-Catalyzed Hydroxylation and THP-Induced Oxidative Rearrangement via QM\/MM Calculations"],"prefix":"10.3390","volume":"31","author":[{"given":"Zheng","family":"Ruan","sequence":"first","affiliation":[{"name":"Shandong Huameng Traditional Chinese Medicine Research Co., Ltd., Jinan 250002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0638-5186","authenticated-orcid":false,"given":"Hong","family":"Li","sequence":"additional","affiliation":[{"name":"College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1686-8272","authenticated-orcid":false,"given":"Yongjun","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xianghui","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-3345-5334","authenticated-orcid":false,"given":"Xinyi","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Chinese Materia Medica Chemistry, Shandong Academy of Chinese Medicine, Jinan 250013, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1016\/j.mam.2004.06.006","article-title":"Carnitine: A nutritional, biosynthetic, and functional perspective","volume":"25","author":"Steiber","year":"2004","journal-title":"Mol. 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