Bhat Saleem Yousuf, Qureshi Insaf Ahmed
Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C.R. Rao Road, Hyderabad 500046, India.
ACS Omega. 2021 Jul 13;6(29):19076-19085. doi: 10.1021/acsomega.1c02386. eCollection 2021 Jul 27.
A leucine aminopeptidase primarily hydrolyzes amino acid leucine from the N-terminus end of proteins and is involved in free amino acid regulation, which makes it a potential therapeutic target against neglected tropical diseases including leishmaniasis. We here report the purification and characterization of the leucine aminopeptidase from (LAP). Using a set of biophysical and biochemical methods, we demonstrate that this enzyme was properly folded after expression in a bacterial system and catalytically active when supplemented with divalent metal cofactors with synthetic fluorogenic peptides. Subsequently, enzymatic inhibition assay denoted that LAP activity was inhibited by peptidomimetics, particularly actinonin, which caused potent inhibition and exhibited stronger binding association with the LAP. Stronger association of actinonin with the LAP was due to a stable complex formation mostly mediated by hydrogen bonding with catalytic and substrate-binding residues in the C-terminal catalytic domain. With molecular dynamics simulation studies, we demonstrate that peptidomimetics retain their topological space in the LAP catalytic pocket and form a stable complex. These results expand the current knowledge of aminopeptidase biochemistry and highlight that specific actinonin or peptidomimetic-based inhibitors may emerge as leads to combat leishmaniasis.
亮氨酸氨肽酶主要从蛋白质的N末端水解氨基酸亮氨酸，并参与游离氨基酸的调节，这使其成为对抗包括利什曼病在内的被忽视热带病的潜在治疗靶点。我们在此报告了来自[具体来源未给出]的亮氨酸氨肽酶（LAP）的纯化和特性。使用一系列生物物理和生化方法，我们证明该酶在细菌系统中表达后正确折叠，并且在补充二价金属辅因子和合成荧光肽时具有催化活性。随后，酶抑制试验表明LAP活性受到拟肽的抑制，特别是放线菌素，它引起了强力抑制并与LAP表现出更强的结合亲和力。放线菌素与LAP的更强结合是由于形成了稳定的复合物，主要由与C末端催化结构域中的催化和底物结合残基的氢键介导。通过分子动力学模拟研究，我们证明拟肽在LAP催化口袋中保留其拓扑空间并形成稳定的复合物。这些结果扩展了当前对氨肽酶生物化学的认识，并突出表明特定的放线菌素或基于拟肽的抑制剂可能成为对抗利什曼病的先导药物。
