Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase
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...
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2025-06-01
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| author | Xiabin Chen Yishuang Li Jianzhuang Yao Xiaoxuan Li Hualing Li Zelin Wu Qi Hu Nuo Xu Tingjun Hou Jiye Wang Shurong Hou |
| author_facet | Xiabin Chen Yishuang Li Jianzhuang Yao Xiaoxuan Li Hualing Li Zelin Wu Qi Hu Nuo Xu Tingjun Hou Jiye Wang Shurong Hou |
| author_sort | Xiabin Chen |
| collection | DOAJ |
| description | 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 <i>E. coli</i> aminopeptidase N (eAPN) was recombinantly produced, and its biochemical and functional profiles were investigated. eAPN’s 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’s 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 > Arg >> Met, Gly > Leu > 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. |
| format | Article |
| id | doaj-art-e74624ab67744aef840e84287ea68fb0 |
| institution | Kabale University |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj-art-e74624ab67744aef840e84287ea68fb02025-08-20T03:26:25ZengMDPI AGBiomolecules2218-273X2025-06-0115682210.3390/biom15060822Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine HydrolaseXiabin Chen0Yishuang Li1Jianzhuang Yao2Xiaoxuan Li3Hualing Li4Zelin Wu5Qi Hu6Nuo Xu7Tingjun Hou8Jiye Wang9Shurong Hou10School of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaSchool of Biological Science and Technology, University of Jinan, Jinan 250022, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, ChinaCollege of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, ChinaKey Laboratory of Drug Prevention and Control Technology of Zhejiang Province, Zhejiang Police College, Hangzhou 310053, ChinaSchool of Pharmacy, Hangzhou Normal University, Hangzhou 311121, China6-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 <i>E. coli</i> aminopeptidase N (eAPN) was recombinantly produced, and its biochemical and functional profiles were investigated. eAPN’s 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’s 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 > Arg >> Met, Gly > Leu > 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.https://www.mdpi.com/2218-273X/15/6/822<i>Escherichia coli</i> aminopeptidase Nheroin and 6-MAMbiochemical propertycatalytic function |
| spellingShingle | Xiabin Chen Yishuang Li Jianzhuang Yao Xiaoxuan Li Hualing Li Zelin Wu Qi Hu Nuo Xu Tingjun Hou Jiye Wang Shurong Hou Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase Biomolecules <i>Escherichia coli</i> aminopeptidase N heroin and 6-MAM biochemical property catalytic function |
| title | Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase |
| title_full | Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase |
| title_fullStr | Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase |
| title_full_unstemmed | Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase |
| title_short | Biochemical and Functional Characterization of <i>E. coli</i> Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase |
| title_sort | biochemical and functional characterization of i e coli i aminopeptidase n a new role as a 6 monoacetylmorphine hydrolase |
| topic | <i>Escherichia coli</i> aminopeptidase N heroin and 6-MAM biochemical property catalytic function |
| url | https://www.mdpi.com/2218-273X/15/6/822 |
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