Harrison P T, Scott J E, Hutchinson M J, Thompson R
Institute of Virology, University of Glasgow, Scotland.
Eur J Biochem. 1995 Jun 1;230(2):511-6. doi: 10.1111/j.1432-1033.1995.0511h.x.
We have constructed a series of mutants to study the role of two structurally and functionally important regions of thymidylate synthase (TS) from varicella-zoster virus (VZV). The first centres on a conserved glycine residue in the beta-kink of beta-strand i, a partially buried region of the protein that is important for dimer interactions and the formation of the active site. We show that the glycine residue located in beta-strand i is not essential for enzyme activity and that beta-strand i can readily accommodate several amino acid substitutions and also an insertion. A covariant residue that accommodates these changes was also identified. The second region of interest was the solvent-exposed and highly mobile C-terminal residue which is an essential component of the active site in TS from Lactobacillus casei and Escherichia coli. We demonstrate that removal of the C-terminal residue from VZV TS does not completely inactivate the enzyme, implying that there are significant structural differences between the virus and bacterial enzymes. By combining site-directed mutagenesis and molecular modelling we have identified these differences and propose a model that explains the contrasting activities.
我们构建了一系列突变体，以研究水痘带状疱疹病毒（VZV）胸苷酸合成酶（TS）两个在结构和功能上重要区域的作用。第一个区域围绕β链i的β-转角处一个保守的甘氨酸残基，该区域是蛋白质的部分埋藏区域，对二聚体相互作用和活性位点的形成很重要。我们发现位于β链i的甘氨酸残基对酶活性并非必需，并且β链i能够轻易容纳多个氨基酸取代以及一个插入。还鉴定出了一个适应这些变化的协变残基。另一个感兴趣的区域是溶剂暴露且高度可变的C末端残基，它是干酪乳杆菌和大肠杆菌TS活性位点的重要组成部分。我们证明从VZV TS中去除C末端残基并不会使酶完全失活，这意味着病毒酶和细菌酶之间存在显著的结构差异。通过结合定点诱变和分子建模，我们确定了这些差异，并提出了一个解释不同活性的模型。
