Abstract：
Pyruvate kinase (PKLR) is a potential candidate gene for milk production traits in cows. The main aim of this work is to investigate the potentially deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) in the PKLR gene by using several computational tools. In silico tools including SIFT, Polyphen-2, SNAP2 and Panther indicated only 18 nsSNPs out of 170 were considered deleterious. The analysis of proteins\' stability change due to amino acid substitution performed by the use of the I-mutant, MUpro, CUPSTAT, SDM and Dynamut confirmed that 9 nsSNPs decreased protein stability. ConSurf analysis predicted that all 18 nsSNPs were evolutionary moderately or highly conserved. Two different domains of PKLR protein were revealed by the InterPro tool with 12 nsSNPs positioned in the Pyruvate Kinase barrel domain and 6 nsSNP present in the Pyruvate Kinase C Terminal. The PKLR 3D model was predicted by MODELLER software and validated via Ramachandran plot and Prosa which indicated a good quality model. The analysis of energy minimizations for the native and mutated structures was performed by SWISS PDB viewer with GROMOS 96 program and showed that 3 structural and 4 functional residues had total energy higher than the native model. These findings indicate that these mutant structures (rs441424814, rs449326723, rs476805413, rs472263384, rs474320860, rs475521477, rs441633284) were less stable than the native model. Molecular Dynamics simulations were performed to confirm the impact of nsSNPs on the protein structure and function. The present study provides useful information about functional SNPs that have an impact on PKLR protein in cattle.Communicated by Ramaswamy H. Sarma.
摘要：
丙酮酸激酶(PKLR)是奶牛产奶性状的潜在候选基因。这项工作的主要目的是通过使用几种计算工具来研究PKLR基因中潜在的有害非同义单核苷酸多态性（nsSNP）。在silico工具，包括SIFT，Polyphen-2，SNAP2和Panther表明170个中只有18个nsSNP被认为是有害的。通过使用I-突变体进行的氨基酸取代导致蛋白质稳定性变化的分析，MUpro,CUPSTAT,SDM和Dynamut证实9个nsSNPs降低了蛋白质的稳定性。ConSurf分析预测所有18个nsSNP是进化中度或高度保守的。通过InterPro工具揭示了PKLR蛋白的两个不同结构域，其中12个nsSNP位于丙酮酸激酶桶结构域中，6个nsSNP位于丙酮酸激酶C末端。通过MODELLER软件预测PKLR3D模型，并通过Ramachandran绘图和Prosa进行验证，表明模型质量良好。通过SWISSPDB查看器使用GROMOS96程序对天然和突变结构进行能量最小化分析，并显示3个结构残基和4个功能残基的总能量高于天然模型。这些发现表明这些突变结构(rs441424814、rs449326723、rs476805413、rs472263384、rs474320860、rs475521477、rs441633284)比天然模型更不稳定。进行分子动力学模拟以证实nsSNP对蛋白质结构和功能的影响。本研究提供了有关对牛PKLR蛋白有影响的功能性SNP的有用信息。由RamaswamyH.Sarma沟通。
