title: Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.
abstract: The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in the trp A gene. Screening of the resulting clones allowed selection of non-interactive mutant alpha subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: alpha 126 valine (GTG)----glutamic acid (GAG) and alpha 128 valine (GTT)----aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as alpha 66 asparagine (AAC)----aspartic acid (GAC); alpha 109 lysine (AAA)----arginine (AGA); alpha 118 cysteine (TGC)----arginine (CGC). Where possible, we individually assessed the importance of these residues in alpha beta interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.
authors.affiliation: Department of Applied Biochemistry and Food Science, University of Nottingham Faculty of Agriculture and Food Sciences, Sutton Bonington, Leicestershire, UK.
authors.forename: S
authors.initials: S
authors.lastname: Swift
authors.forename: J
authors.initials: J
authors.lastname: Kuhn
authors.forename: G S
authors.initials: GS
authors.lastname: Stewart
mesh_terms: D000596:Amino Acids; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics*
chemical_list: D000596:Amino Acids; D014367:Tryptophan Synthase