# Biochemical and Functional Characterization of E. coli Aminopeptidase N: A New Role as a 6-Monoacetylmorphine Hydrolase

## TL;DR

This paper identifies E. coli aminopeptidase N as a potential enzyme for breaking down heroin metabolites, offering new insights for detoxification.

## Contribution

The study reveals E. coli aminopeptidase N's novel ability to hydrolyze 6-monoacetylmorphine, a key heroin metabolite.

## Key findings

- eAPN shows optimal activity at pH 7.5 and is thermostable.
- eAPN preferentially cleaves peptides with small or basic amino acid residues.
- eAPN can hydrolyze 6-MAM, suggesting potential for heroin detoxification.

## Abstract

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’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.