title: Site-directed mutagenesis of varicella-zoster virus thymidylate synthase. Analysis of two highly conserved regions of the enzyme.
abstract: 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.
authors.affiliation: Institute of Virology, University of Glasgow, Scotland.
authors.forename: P T
authors.initials: PT
authors.lastname: Harrison
authors.forename: J E
authors.initials: JE
authors.lastname: Scott
authors.forename: M J
authors.initials: MJ
authors.lastname: Hutchinson
authors.forename: R
authors.initials: R
authors.lastname: Thompson
mesh_terms: D001483:Base Sequence; D017124:Conserved Sequence; D014645:Herpesvirus 3, Human / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics*
chemical_list: D009838:Oligodeoxyribonucleotides; D013940:Thymidylate Synthase