Kumar Pravin, Ghosh Sachan Shashwati, Poddar Raju
Department of Bio-Engineering, Birla Institute of Technology-Mesra, Ranchi, JH, 835 215, India.
Department of Bio-Engineering, Birla Institute of Technology-Mesra, Ranchi, JH, 835 215, India.
J Mol Graph Model. 2017 Oct;77:94-105. doi: 10.1016/j.jmgm.2017.08.014. Epub 2017 Aug 18.
Improving the industrial enzyme for better yield of the product is important and a challenging task. One of such important industrial enzymes is microbial Hydroxycinnamoyl-CoA hydratase-lyase (HCHL). It converts feruloyl-CoA to vanillin. We place our efforts towards the improvement of its catalytic activity with comprehensive computational investigation. Catalytic core of the HCHL was explored with molecular modeling and docking approaches. Site-directed mutations were introduced in the catalytic site of HCHL in a sequential manner to generate different mutants of HCHL. Basis of mutation is to increase the interaction between HCHL and substrate feruloyl-CoA through interatomic forces and hydrogen bond formation. A rigorous molecular dynamics (MD) simulation was performed to check the stability of mutant's structure. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), dynamic cross correlation (DCCM) and principal component analysis (PCA) were also performed to analyze flexibility and stability of structures. Docking studies were carried out between different mutants of HCHL and feruloyl-CoA. Investigation of the different binding sites and the interactions with mutant HCHLs and substrate allowed us to highlight the improved performance of mutants than wild type HCHL. This was further validated with MD simulation of complex consisting of different mutants and substrate. It further confirms all the structures are stable. However, mutant-2 showed better affinity towards substrate by forming hydrogen bond between active site and feruloyl-CoA. We propose that increase in hydrogen bond formation might facilitate in dissociation of vanillin from feruloyl-CoA. The current work may be useful for the future development of 'tailor-made' enzymes for better yield of vanillin.
提高工业酶以获得更高的产品产量是一项重要且具有挑战性的任务。微生物羟基肉桂酰辅酶A水合酶裂解酶（HCHL）就是这样一种重要的工业酶。它能将阿魏酰辅酶A转化为香草醛。我们通过全面的计算研究致力于提高其催化活性。采用分子建模和对接方法探索了HCHL的催化核心。以序列方式在HCHL的催化位点引入定点突变以产生不同的HCHL突变体。突变的依据是通过原子间力和氢键形成增加HCHL与底物阿魏酰辅酶A之间的相互作用。进行了严格的分子动力学（MD）模拟以检查突变体结构的稳定性。还进行了均方根偏差（RMSD）、均方根波动（RMSF）、动态交叉相关（DCCM）和主成分分析（PCA）以分析结构的柔韧性和稳定性。对HCHL的不同突变体与阿魏酰辅酶A进行了对接研究。对不同结合位点以及突变型HCHL与底物之间相互作用的研究使我们能够突出突变体比野生型HCHL具有更好的性能。这通过由不同突变体和底物组成的复合物的MD模拟得到了进一步验证。它进一步证实所有结构都是稳定的。然而，突变体2通过在活性位点和阿魏酰辅酶A之间形成氢键表现出对底物更好的亲和力。我们提出氢键形成的增加可能有助于香草醛从阿魏酰辅酶A中解离。目前的工作可能对未来开发用于更好地生产香草醛的“定制”酶有用。
