香兰素在各种行业中作为调味剂具有重要意义,包括食物,制药,和化妆品。从阿魏酸生物合成香草醛的CoA依赖性途径涉及阿魏酸酰辅酶A合酶(Fcs)和烯酰辅酶A水合酶/裂解酶(Ech)。在这项研究中,Fcs和Ech来自链霉菌。菌株V-1。通过包括序列比对和分子动力学模拟在内的计算技术分析了Ech的序列保守性和结构特征。在详细研究了Ech与底物之间的主要结合模式和关键氨基酸残基后,一系列突变(F74W,A130G,A130G/T132S,R147Q,Q255R,ΔT90,ΔTGPEIL,通过合理设计获得ΔN1-11,ΔC260-287)。最后,通过全细胞催化验证了这些突变体产生的香兰素的产量。结果表明,三个突变体,F74W,Q147R,和ΔN1-11显示出比野生型Ech更高的产量。分子动力学模拟和残基能量分解确定了碱性残基K37,R38,K561和R564是影响Ech与阿魏酸辅酶A(FCA)之间结合自由能的关键残基。由突变引起的静电相互作用和极性溶剂化能量的巨大变化可能导致酶活性降低。本研究为香兰素的生物合成途径提供了重要的理论指导和实验数据。
Vanillin holds significant importance as a flavoring agent in various industries, including food, pharmaceuticals, and cosmetics. The CoA-dependent pathway for the biosynthesis of vanillin from ferulic acid involved feruloyl-CoA synthase (Fcs) and enoyl-CoA hydratase/lyase (Ech). In this research, the Fcs and Ech were derived from Streptomyces sp. strain V-1. The sequence conservation and structural features of Ech were analyzed by computational techniques including sequence alignment and molecular dynamics simulation. After detailed study for the major binding modes and key amino acid residues between Ech and substrates, a series of mutations (F74W, A130G, A130G/T132S, R147Q, Q255R, ΔT90, ΔTGPEIL, ΔN1-11, ΔC260-287) were obtained by rational design. Finally, the yield of vanillin produced by these mutants was verified by whole-cell catalysis. The results indicated that three mutants, F74W, Q147R, and ΔN1-11, showed higher yields than wild-type Ech. Molecular dynamics simulations and residue energy decomposition identified the basic residues K37, R38, K561, and R564 as the key residues affecting the free energy of binding between Ech and feruloyl-coenzyme A (FCA). The large changes in electrostatic interacting and polar solvating energies caused by the mutations may lead to decreased enzyme activity. This study provides important theoretical guidance as well as experimental data for the biosynthetic pathway of vanillin.