title: Saturation site-directed mutagenesis of thymidylate synthase.
abstract: We have subjected 12 different codons of a synthetic Lactobacillus casei thymidylate synthase (TS) gene to saturation site-directed mutagenesis to create amino acid "replacement sets" at each of those positions. The target residues were chosen because they are highly conserved and because they are important for the structure and function of the protein as indicated by solution and structural studies. The mutagenesis procedure involved excision of a fragment of the synthetic gene containing the target codon, followed by its replacement with a mixture of oligonucleotides which code for all 20 amino acids and the amber stop codon. TS mutants were identified by DNA sequencing, and catalytically active mutants were identified by genetic complementation using a Thy- strain of Escherichia coli. Only 3 of the 12 target amino acids examined were essential for TS activity; and of the 125 total mutants identified, 57 were catalytically active. These results point to a high degree of plasticity of TS in accommodating function with structural change.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
authors.forename: S
authors.initials: S
authors.lastname: Climie
authors.forename: L
authors.initials: L
authors.lastname: Ruiz-Perez
authors.forename: D
authors.initials: D
authors.lastname: Gonzalez-Pacanowska
authors.forename: P
authors.initials: P
authors.lastname: Prapunwattana
authors.forename: S W
authors.initials: SW
authors.lastname: Cho
authors.forename: R
authors.initials: R
authors.lastname: Stroud
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D000595:Amino Acid Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D013940:Thymidylate Synthase / Q000235:genetics*
chemical_list: D003062:Codon; D013940:Thymidylate Synthase