Elatico Adam Jo J, Nellas Ricky B
Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, Philippines.
Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, Philippines.
J Mol Graph Model. 2020 Nov;100:107657. doi: 10.1016/j.jmgm.2020.107657. Epub 2020 Jun 24.
Lipases are important enzymes in many biochemical industries, thus making them attractive targets for protein engineering to improve enzymatic properties. In this work, a ''reverse engineering'' approach was explored: disrupt secondary structures to determine their contribution to enzyme stability and activity. All the α-helices of the lipase from Pseudomonas aeruginosa PAO1 (PAL) were systematically disrupted using computational proline mutagenesis and molecular dynamics (MD) simulations. This method identified the α3 mutant (R89P), located within the vicinity of the active site, to be significantly important for stability and activity. In addition, the α6 system (L159P), part of the ''cap'' domain that regulates substrate entry into the active site, was found to be critical for activity as it pushed the lipase to adopt a completely closed conformation. The perturbation introduced by the proline mutations resulted in increased backbone flexibility that significantly decreased protein stability. Moreover, mutations within the cap domain helices - α4 (A115P), α5 (S132P, G139P), α6 (L159P), and α7 (R169P) - resulted in increased flexibility of the N-terminal region of the α5 helix, the mobile ''lid'' helix, that pushes the gorge into a partially closed conformation. The α6 mutation (L159P) further increased the flexibility of the helix-loop region at the C-terminal end of the α5 helix to push the lid into the fully closed state. Therefore, the α3 and α6 helices could be ''hot spots'' for stabilizing mutations that could improve the overall enzyme stability and activity this lipase. The insights obtained in this work may be validated experimentally in future works.
脂肪酶是许多生化产业中的重要酶，因此使其成为蛋白质工程改善酶特性的有吸引力的目标。在这项工作中，探索了一种“逆向工程”方法：破坏二级结构以确定它们对酶稳定性和活性的贡献。使用计算脯氨酸诱变和分子动力学（MD）模拟系统地破坏了铜绿假单胞菌PAO1（PAL）脂肪酶的所有α螺旋。该方法确定位于活性位点附近的α3突变体（R89P）对稳定性和活性非常重要。此外，发现作为调节底物进入活性位点的“帽”结构域一部分的α6系统（L159P）对活性至关重要，因为它促使脂肪酶采取完全封闭的构象。脯氨酸突变引入的扰动导致主链灵活性增加，从而显著降低蛋白质稳定性。此外，帽结构域螺旋内的突变——α4（A115P）、α5（S132P、G139P）、α6（L159P）和α7（R169P）——导致α5螺旋（可移动的“盖子”螺旋）N端区域的灵活性增加，将峡谷推至部分封闭的构象。α6突变（L159P）进一步增加了α5螺旋C端螺旋-环区域的灵活性，将盖子推至完全封闭状态。因此，α3和α6螺旋可能是稳定突变的“热点”，可提高这种脂肪酶的整体酶稳定性和活性。这项工作中获得的见解可能在未来的工作中通过实验得到验证。
