Wang Shang, Xu Yan, Yu Xiao-Wei
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PRC.
J Agric Food Chem. 2021 Apr 14;69(14):4263-4275. doi: 10.1021/acs.jafc.1c00721. Epub 2021 Apr 2.
Most fungal lipases contain a propeptide, which is very important for their function and substrate selectivity. In the present study, lipase (RCL) was used as a research model to explore the mechanism of the propeptide of the lipase. Conventional molecular dynamics (MD) and metadynamics simulations were used to explore the mechanism by which the propeptide affects the activity of the lipase, which was subsequently verified by mutation experiments. MD simulations indicated that the propeptide had an inhibitory effect on the lid movement of RCL and found a key region (Val5-Thr10) on the propeptide. Subsequently, site-directed mutations were created in this region. The mutations enhanced the lipase catalytic efficiency to 700% and showed the potential for the propeptide to shift the substrate specificity of RCL. The specificity and activity of RCL mutants also had similar trends to wild-type RCL toward triglycerides with varying chain lengths. The mutual corroboration of simulation and site-directed mutagenesis results revealed the vital role of the key propeptide region in the catalytic activity and substrate specificity of the lipase.
大多数真菌脂肪酶含有一个前肽，这对其功能和底物选择性非常重要。在本研究中，脂肪酶（RCL）被用作研究模型来探索脂肪酶前肽的作用机制。采用传统分子动力学（MD）和元动力学模拟来探究前肽影响脂肪酶活性的机制，随后通过突变实验进行验证。MD模拟表明，前肽对RCL的盖子运动具有抑制作用，并在前肽上发现了一个关键区域（Val5-Thr10）。随后，在该区域进行了定点突变。这些突变将脂肪酶的催化效率提高到了700%，并显示出前肽改变RCL底物特异性的潜力。RCL突变体对不同链长甘油三酯的特异性和活性与野生型RCL也有相似趋势。模拟结果和定点诱变结果的相互印证揭示了关键前肽区域在脂肪酶催化活性和底物特异性中的重要作用。
