{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T06:16:05Z","timestamp":1772000165091,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T00:00:00Z","timestamp":1770940800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Brain Science and Brain-like Intelligence Technology-National Science and Technology Major Project","award":["2022ZD0214600"],"award-info":[{"award-number":["2022ZD0214600"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82073749"],"award-info":[{"award-number":["82073749"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["22273032"],"award-info":[{"award-number":["22273032"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Interdisciplinary Research Project of Hangzhou Normal University","award":["2025JCXK02"],"award-info":[{"award-number":["2025JCXK02"]}]},{"name":"Central Guidance on the Development of Local Science and Technology","award":["2025ZY01109"],"award-info":[{"award-number":["2025ZY01109"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Heroin is a highly addictive drug that exerts its primary effects through activation of \u03bc-opioid receptors. Its principal active metabolite, 6-monoacetylmorphine (6-MAM), significantly contributes to heroin\u2019s neurological effects and acute toxicity. Current pharmacotherapies for heroin use disorder, employing opioid receptor agonist or antagonist, are often limited by risks of dependence, tolerance, and\/or adverse side effects. In this context, enzyme-based therapy emerges as a promising alternative by rapidly converting drugs into inactive or less harmful metabolites in the blood. As a macromolecule, the enzyme does not cross the blood\u2013brain barrier, thereby avoiding side effects in CNS. Through structure-based computational screening, Xoo-PepA (PDB ID: 3JRU), a leucine aminopeptidase from Xanthomonas oryzae pv. oryzae, was identified as a potential enzyme capable of hydrolyzing heroin and 6-MAM. Computational and experimental analyses confirm that Xoo-PepA hydrolyzes heroin sequentially to 6-MAM and subsequently to morphine. Enzymatic properties including dependence on metal ions, optimal pH, thermal stability, and substrate specificity were characterized accordingly. Notably, supplementation with Ni2+ or Zn2+ and TCEP extended Xoo-PepA\u2019s half-life at 37 \u00b0C from 1 h to over 24 h, highlighting the essential role of metal ions in maintaining structural stability. Moreover, Ni2+ enhanced Xoo-PepA\u2019s hydrolysis toward peptidase substrate L-leucine-p-nitroaniline by 770-fold, yet conferred no significant activation toward heroin. Mutations in metal ion-coordination residues (e.g., K262A, D267A\/E346L) exhibited different activity profiles toward these two types of substrates, suggesting a distinct regulatory mechanism of metal ions may be involved in these activities. This study provides the first demonstration that Xoo-PepA, a non-mammalian, metal-dependent aminopeptidase, can hydrolyze heroin and 6-MAM, shedding light on its functional versatility and biochemical characteristics.<\/jats:p>","DOI":"10.3390\/biom16020298","type":"journal-article","created":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T16:09:32Z","timestamp":1770998972000},"page":"298","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Leucine Aminopeptidase from Xanthomonas oryzae pv. oryzae with Esterase Activity Toward Heroin: Biochemical and Catalytic Insights"],"prefix":"10.3390","volume":"16","author":[{"given":"Hualing","family":"Li","sequence":"first","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qi","family":"Hu","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nuo","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xueting","family":"Shao","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuxin","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuxin","family":"Hou","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Binjie","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Drug Prevention and Control Technology of Zhejiang Province, Zhejiang Police College, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiye","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Drug Prevention and Control Technology of Zhejiang Province, Zhejiang Police College, Hangzhou 310053, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5017-3677","authenticated-orcid":false,"given":"Jianzhuang","family":"Yao","sequence":"additional","affiliation":[{"name":"School of Biological Science and Technology, University of Jinan, Jinan 250022, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shurong","family":"Hou","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiabin","family":"Chen","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"United Nations Office on Drugs and Crime (2025). 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