Functional characterization of hypothetical proteins of <i>Mycobacterium tuberculosis</i> with possible esterase/lipase signature: a cumulative <i>in silico</i> and <i>in vitro</i> approach

The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, <i>in silico</i> approaches were used to predict the biological function of some of the unknown <i>Mtb</i> proteins, which were further validated by wet lab experiments. After screening thousands of <i>Mtb</i> proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c, and Rv3591c were chosen on the basis of their importance in <i>Mtb</i> life cycle. All these proteins posses the α/β-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif “GXSXG” and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin and phenylmethanesulfonyl fluoride (PMSF)). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the <i>in silico</i> studies, these genes were cloned from <i>Mtb</i> genome and the proteins were over-expressed in pQE-30/<i>Escherichia coli</i> M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyze and characterize unknown hypothetical proteins.多种被注释为假想蛋白的分枝杆菌蛋白，其功能仍有待揭示。在本研究中，我们采用计算生物学（in silico）方法预测了部分未知的结核分枝杆菌（Mtb）蛋白的生物学功能，并通过湿实验室实验对预测结果进行了验证。在筛选了数千株结核分枝杆菌蛋白后，我们基于这些蛋白在结核分枝杆菌生命周期中的重要性，挑选出了功能未知的假想蛋白Rv0421c、Rv0519c、Rv0774c、Rv1191、Rv1592c及Rv3591c。上述所有蛋白均具有α/β水解酶拓扑折叠结构，这是脂肪酶/酯酶的典型特征，其催化三联体的推定氨基酸残基为丝氨酸、天冬氨酸和组氨酸。催化丝氨酸位于五肽基序"GXSXG"中，而氧阴离子残基则位于二肽基序HG中。为进一步佐证我们的发现，我们采用常规合成的脂解底物（对硝基苯酚酯类，pNP-esters）以及特异性脂肪酶/酯酶抑制剂（四氢脂抑素（tetrahydrolipstatin）和苯甲基磺酰氟（PMSF））进行了分子对接实验。底物与抑制剂和这些蛋白的对接得分显著且相互作用强烈，表明它们极有可能为脂肪酶/酯酶。为验证计算生物学研究结果，我们从结核分枝杆菌基因组中克隆了上述基因，并在pQE-30/大肠杆菌（Escherichia coli）M15表达系统中对目标蛋白进行了过表达。将表达的蛋白纯化至均一状态，并以对硝基苯酚酯类为底物测定了其酶活性。重组蛋白的酶活性可被四氢脂抑素和苯甲基磺酰氟预处理所抑制。本研究的结果为未知假想蛋白的分析与功能鉴定提供了基础平台。

搜集汇总

背景与挑战

背景概述

该数据集聚焦于结核分枝杆菌中具有酯酶/脂肪酶特征的假设蛋白的功能表征，采用计算（in silico）和实验（in vitro）相结合的方法进行研究。通过筛选和验证6个关键蛋白（如Rv0421c等），发现它们具有α/β-水解酶拓扑折叠和催化三联体特征，分子对接和酶活性实验支持其酯酶/脂肪酶功能。这为未知蛋白的功能分析提供了基础平台，数据集涵盖生物化学、微生物学等多个类别，关键词包括结核分枝杆菌、假设蛋白和分子对接等。