Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.
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.
Swift
S
S
Department of Applied Biochemistry and Food Science, University of Nottingham Faculty of Agriculture and Food Sciences, Sutton Bonington, Leicestershire, UK.
Kuhn
J
J
Stewart
G S
GS
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*
D000596:Amino Acids; D014367:Tryptophan Synthase