Journal Article

Leucine Aminopeptidase from Xanthomonas oryzae pv. oryzae with Esterase Activity Toward Heroin: Biochemical and Catalytic Insights

Xu, Nuo, Shao, Xueting, Wang, Jiye, Yao, Jianzhuang, Wang, Binjie, Liu, Yuxin, Li, Hualing, Hou, Yuxin, Hou, Shurong, Chen, Xiabin, Hu, Qi

2026

Overview

Heroin is a highly addictive drug that exerts its primary effects through activation of μ-opioid receptors. Its principal active metabolite, 6-monoacetylmorphine (6-MAM), significantly contributes to heroin’s 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–brain 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’s half-life at 37 °C from 1 h to over 24 h, highlighting the essential role of metal ions in maintaining structural stability. Moreover, Ni2+ enhanced Xoo-PepA’s 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.

• Publisher: MDPI AG,Multidisciplinary Digital Publishing Institute (MDPI)
• Subject: Acute toxicity / Aminopeptidase / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biochemical characteristics / biochemical characterization / Biotechnology / Blood-brain barrier / catalytic function / Chromatography / Cocaine / Drug abuse / Drug overdose / E coli / Enzymes / Heroin / Heroin - chemistry / Heroin - metabolism / heroin abuse / Hydrogen-Ion Concentration / Hydrolysis / Leucine / Leucyl Aminopeptidase - chemistry / Leucyl Aminopeptidase - genetics / Leucyl Aminopeptidase - metabolism / Metabolism / Metabolites / Metal ions / Morphine / Narcotics / Opioid receptors / Physiology / Plasmids / Proteins / Side effects / Substance abuse treatment / Substrate Specificity / Thermal stability / Toxicity / Xanthomonas - enzymology / Xanthomonas oryzae / Zinc