Reshma S V, Sathyanarayanan Nitish, Nagendra H G
Department of Biotechnology, PES University, Bangalore.
Department of Biotechnology, Sir M Visvesvaraya Institute of Technology, Hunasemaranahalli, Bangalore 562157.
Bioinformation. 2017 Dec 31;13(12):430-438. doi: 10.6026/97320630013430. eCollection 2017.
Hypothetical proteins are functionally uncharacterized proteins with assigned function using sequence annotation tools. Almost half of the coding regions of several genomes are hypothetical proteins. Therefore, it is of our interest to characterize a hypothetical protein YVRE from the model system Bacillus subtilis using known data. YVRE is assigned the function as a glucono-lactonase using prediction and phylogenetic analysis. A molecular dynamics simulated homology model of YVRE (with calcium) using human senescence marker protein 30 /SMP30 (PDB ID: 3G4E) as template is reported for functional inference. It is observed that the protein possesses bivalent metal binding domain. Molecular docking studies with the substrate glucono-δ-lactone show YVRE binding with the substrate. This data was further validated using cloning and sub-cloning in pUC57 and pET22b+ respectively, followed by expression and purification using nickel affinity chromatography. The activity of YVRE using the substrate glucono-δ-lactone was calculated. The results show the function of YVRE as a gluconolactonase, with higher preference to zinc than calcium or magnesium. Thus, YVRE is shown to play key role in three metabolic pathways namely, pentose phosphate pathway, ascorbate and aldarate metabolism, and caprolactam degradation.
假设蛋白是通过序列注释工具进行功能注释但功能尚未明确的蛋白。几个基因组中几乎一半的编码区域都是假设蛋白。因此，利用已知数据对模式系统枯草芽孢杆菌中的一个假设蛋白YVRE进行功能表征引起了我们的兴趣。通过预测和系统发育分析，YVRE被赋予葡萄糖酸内酯酶的功能。本文报道了一个以人类衰老标记蛋白30/SMP30（PDB ID：3G4E）为模板的YVRE（含钙）分子动力学模拟同源模型，用于功能推断。观察到该蛋白具有二价金属结合结构域。与底物葡萄糖酸-δ-内酯的分子对接研究表明YVRE与底物结合。分别在pUC57和pET22b+中进行克隆和亚克隆，随后使用镍亲和层析进行表达和纯化，进一步验证了该数据。计算了YVRE使用底物葡萄糖酸-δ-内酯的活性。结果表明YVRE具有葡萄糖酸内酯酶的功能，对锌的偏好高于钙或镁。因此，YVRE在磷酸戊糖途径、抗坏血酸和醛糖代谢以及己内酰胺降解这三条代谢途径中发挥关键作用。
