{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T01:31:21Z","timestamp":1771983081642,"version":"3.50.1"},"reference-count":41,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,6,5]],"date-time":"2025-06-05T00:00:00Z","timestamp":1749081600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"STI2030-Major Projects","award":["2022ZD0214600"],"award-info":[{"award-number":["2022ZD0214600"]}]},{"name":"STI2030-Major Projects","award":["82073749"],"award-info":[{"award-number":["82073749"]}]},{"name":"STI2030-Major Projects","award":["22273032"],"award-info":[{"award-number":["22273032"]}]},{"name":"STI2030-Major Projects","award":["2025JCXK02"],"award-info":[{"award-number":["2025JCXK02"]}]},{"name":"National Natural Science Foundation of China","award":["2022ZD0214600"],"award-info":[{"award-number":["2022ZD0214600"]}]},{"name":"National Natural Science Foundation of China","award":["82073749"],"award-info":[{"award-number":["82073749"]}]},{"name":"National Natural Science Foundation of China","award":["22273032"],"award-info":[{"award-number":["22273032"]}]},{"name":"National Natural Science Foundation of China","award":["2025JCXK02"],"award-info":[{"award-number":["2025JCXK02"]}]},{"name":"Interdisciplinary Research Project of Hangzhou Normal University","award":["2022ZD0214600"],"award-info":[{"award-number":["2022ZD0214600"]}]},{"name":"Interdisciplinary Research Project of Hangzhou Normal University","award":["82073749"],"award-info":[{"award-number":["82073749"]}]},{"name":"Interdisciplinary Research Project of Hangzhou Normal University","award":["22273032"],"award-info":[{"award-number":["22273032"]}]},{"name":"Interdisciplinary Research Project of Hangzhou Normal University","award":["2025JCXK02"],"award-info":[{"award-number":["2025JCXK02"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>6-monoacetylmorphine (6-MAM), a primary active metabolite of heroin that reaches the human brain, plays a crucial role in producing heroin-associated physiological and lethal effects. Therefore, 6-MAM has emerged as a key target for alleviating the adverse consequences of heroin abuse. In this study, the proposed 6-MAM hydrolase E. coli aminopeptidase N (eAPN) was recombinantly produced, and its biochemical and functional profiles were investigated. eAPN\u2019s biochemical properties, with respect to pH, metal ions, and temperature, and catalytic functions toward peptidase substrates and 6-MAM were thoroughly examined. Extensive experiments reveal that incorporation of an N-terminal His-tag notably affects eAPN\u2019s aminopeptidase activity. This cost-effective recombinant eAPN exhibits favorable thermostability and optimal activity at pH 7.5. Kinetic analysis toward peptidase substrates reveals that eAPN preferentially cleaves peptides following amino acid residues in the order of Ala &gt; Arg &gt;&gt; Met, Gly &gt; Leu &gt; Pro, indicating a preference for small or basic amino acid residues as substrates. Computational and experimental studies have, for the first time, discovered that eAPN is capable of catalyzing the hydrolysis of heroin and 6-MAM, which has shed light on its functional versatility and potential applications. This work elucidates the biochemical properties of eAPN and expands its catalytic functions, thereby laying the groundwork for a deep understanding and further reengineering of eAPN to enhance its activity toward 6-MAM for heroin detoxification.<\/jats:p>","DOI":"10.3390\/biom15060822","type":"journal-article","created":{"date-parts":[[2025,6,5]],"date-time":"2025-06-05T09:43:18Z","timestamp":1749116598000},"page":"822","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Biochemical and Functional Characterization of E. coli Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase"],"prefix":"10.3390","volume":"15","author":[{"given":"Xiabin","family":"Chen","sequence":"first","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yishuang","family":"Li","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, 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":"Xiaoxuan","family":"Li","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hualing","family":"Li","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zelin","family":"Wu","sequence":"additional","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":"Tingjun","family":"Hou","sequence":"additional","affiliation":[{"name":"College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, 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"}]},{"given":"Shurong","family":"Hou","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"109","DOI":"10.2174\/157488406775268219","article-title":"Pharmacokinetics and pharmacokinetic variability of heroin and its metabolites: Review of the literature","volume":"1","author":"Rook","year":"2006","journal-title":"Curr. 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