Gaur Neha, Kumar Kishore, Gupta Chhedi Lal, Saxena Jitendra Kumar
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India.
Division of Biochemistry, Regional Research Institute of Unani Medicine, Bhadrak, 756100, Odisha, India.
Protein Expr Purif. 2019 Jul;159:1-9. doi: 10.1016/j.pep.2019.02.018. Epub 2019 Mar 1.
Arginase catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and hence potential drug target for the treatment of leishmaniasis. The arginase from Leishmania donovani (LdARG) was cloned, overexpressed and characterized. Analysis of the deduced amino acid sequence of LdARG with homologous enzyme from other trypanosomatids arginases identified a non-conserved 12 residues long segment VWGLIERTFLSA from position 161-172. This counter segment in L. mexicana arginase exhibits a different conformation compared with human arginase I. The pH and temperature optima of LdARG were 9.0 and 37 °C, respectively. Biochemical studies revealed that the K for the substrate L-arginine was 24.76 ± 0.06 mM. Molecular modeling of LdARG studies revealed that the glutamic acid residue at position 288 plays a role in substrate binding. The importance of this glutamic acid residue was validated by constructing a mutant variant of LdARG (E288Q-LdARG) by replacing glutamic acid with glutamine through site-directed mutagenesis. The K value of mutant variant for L-arginine was found to be 107 ± 0.18 mM. The increase in K value of E288Q-LdARG as compared to LdARG suggested that substrate binding was significantly affected which could be exploited further. Studies on biochemical and structural characterization of recombinant LdARG will help in evaluating this enzyme as a potential drug target for visceral leishmaniasis.
精氨酸酶催化多胺生物合成中的首个关键步骤，多胺能促进细胞生长，因此是治疗利什曼病的潜在药物靶点。杜氏利什曼原虫的精氨酸酶（LdARG）被克隆、过量表达并进行了特性分析。将LdARG推导的氨基酸序列与其他锥虫精氨酸酶的同源酶进行分析，确定了一个位于161 - 172位的12个残基长的非保守片段VWGLIERTFLSA。墨西哥利什曼原虫精氨酸酶中的这个对应片段与人类精氨酸酶I相比呈现出不同的构象。LdARG的最适pH和温度分别为9.0和37°C。生化研究表明，其底物L - 精氨酸的K值为24.76±0.06 mM。LdARG的分子模拟研究表明，288位的谷氨酸残基在底物结合中起作用。通过定点诱变将谷氨酸替换为谷氨酰胺构建LdARG的突变体变体（E288Q - LdARG），验证了这个谷氨酸残基的重要性。发现突变体变体对L - 精氨酸的K值为107±0.18 mM。与LdARG相比，E288Q - LdARG的K值增加表明底物结合受到显著影响，这一点可进一步利用。对重组LdARG的生化和结构特性研究将有助于评估这种酶作为内脏利什曼病潜在药物靶点的可能性。
