Shahab Mohd, Verma Meenakshi, Pathak Manisha, Mitra Kalyan, Misra-Bhattacharya Shailja
Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India; Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
PLoS One. 2014 Jun 18;9(6):e99884. doi: 10.1371/journal.pone.0099884. eCollection 2014.
Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (Km) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.
沃尔巴克氏体是丝虫线虫的一种内共生菌，被认为是治疗淋巴丝虫病的一个有前景的靶点。尽管对沃尔巴克氏体肽聚糖组装的功能特性尚未进行充分研究，但沃尔巴克氏体基因组为编码肽聚糖生物合成途径中脂质II生物合成所涉及的所有基因提供了证据。UDP-N-乙酰葡糖胺烯醇丙酮酸转移酶（MurA）是脂质II生物合成途径中的一种酶，不可避免地被视为一种抗生素靶点。鉴于MurA在细菌生存能力和存活中的关键作用，克隆、表达并纯化了来自马来布鲁线虫沃尔巴克氏体内共生菌的MurA直系同源物（wBm-MurA），以进行进一步的分子特性分析。使用磷霉素进行了酶动力学和抑制研究。发现wBm-MurA在马来布鲁线虫的所有主要生命阶段均有表达，并通过共聚焦显微镜在微丝蚴和雌性成虫体内的沃尔巴克氏体中进行了免疫定位。序列分析表明，对酶活性至关重要的氨基酸是保守的。纯化后的wBm-MurA在较宽的pH范围内具有类似EPSP合酶（3-磷酸莽草酸1-羧乙烯基转移酶）的活性，在pH 7.5和37°C温度下活性最佳。发现底物UDP-N-乙酰葡糖胺的表观亲和力常数（Km）为0.03149 mM，磷酸烯醇丙酮酸的表观亲和力常数为0.009198 mM。在磷霉素存在的情况下，相对酶活性被抑制了约2倍。wBm-MurA同源模型与流感嗜血杆菌（Hi-MurA）结构模型的叠加表明磷霉素在相同的活性位点结合。这些发现表明wBm-MurA是筛选新型化合物的一个推定抗丝虫药物靶点。
