Department of Biochemistry, BMS Block 2, South Campus, Panjab University, Sector 25, Chandigarh 160014, India.
Department of Biotechnology, BMS Block 1, South Campus, Panjab University, Sector 25, Chandigarh 160014, India.
J Med Microbiol. 2019 Nov;68(11):1629-1640. doi: 10.1099/jmm.0.001080.
ML1899 is conserved in all mycobacterium sp. and is a middle member of operon involved in mycolic acid modification. In the present study attempts were made to characterize ML1899 in detail.. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA. analysis predicted ML1899 as a member of / hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM min catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. , and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min ml and 27 U mg , respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.
ML1899 在所有分枝杆菌属中都保守存在，是参与类脂酸修饰的操纵子的中间成员。本研究试图详细表征 ML1899。使用生物信息学工具预测活性位点残基、抗原表位和蛋白质的三维模型。该基因被克隆、表达和纯化为 His 标记的蛋白，用于生物物理/生物化学特性分析。重组蛋白用于处理 THP-1 细胞，通过流式细胞术/ELISA 研究一氧化氮（NO）、活性氧（ROS）、细胞因子和趋化因子产生的变化。结构分析预测 ML1899 为 /水解酶家族成员，具有 GXSXG 基序和 Ser126、His282、Asp254 作为活性位点残基，通过定点诱变得到证实。ML1899 表现出酯酶活性。它以 pNP-丁酸为最佳底物，在 pH8.0 和 50°C 下具有 5.56µM min 的催化效率。该酶在 60°C 温度和 pH6.0 至 9.0 之间具有稳定性。计算出 ML1899 的酶活力 Km 值、最适 pH 值和比活力分别为 400µM、40µmoles min ml 和 27U mg 。ML1899 还表现出磷脂酶活性。当以较高浓度处理时，该蛋白会影响巨噬细胞的存活。ML1899 增强了 ROS/NO 的产生，并上调了促炎细胞因子和趋化因子，包括 TNF-α、IFN-γ、IL-6 和 IL-8 在巨噬细胞中的表达。还观察到 ML1899 在 69%的麻风病患者中引起体液反应。这些结果表明，ML1899 作为一种酯酶，在低浓度时可以上调有利于巨噬细胞的免疫反应，但在高浓度时会杀死 THP-1 巨噬细胞。
