DNA sequence analysis of artificially evolved ebg enzyme and ebg repressor genes.
The ebg system has been used as a model to study the artificial selection of new catalytic functions of enzymes and of inducer specificities of repressors. A series of mutant enzymes with altered catalytic specificities were previously characterized biochemically as were the changes in inducer specificities of mutant, but fully functional, repressors. The wild type ebg operon has been sequenced, and the sequence differences of the mutant enzymes and repressors have been determined. We now report that, contrary to our previous understanding, ebg enzyme contains 180-kD alpha-subunits and 20-kD beta-subunits, both of which are required for full activity. Mutations that dramatically affect substrate specificity and catalytic efficiency lie in two distinct regions, both well outside of the active site region. Mutations that affect inducer specificity of the ebg repressor lie within predicted sugar binding domains. Comparisons of the ebg beta-galactosidase and repressor with homologous proteins of the Escherichia coli and Klebsiella pneumoniae lac operons, and with the galactose operon repressor, suggest that the ebg and lac operons diverged prior to the divergence of E. coli from Klebsiella. One case of a triple substitution as the consequence of a single event is reported, and the implications of that observation for mechanisms of spontaneous mutagenesis are discussed.
Hall
B G
BG
Molecular and Cell Biology, University of Connecticut, Storrs 06268.
Betts
P W
PW
Wootton
J C
JC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D005075:Biological Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005696:Galactosidases / Q000235:genetics*; D005784:Gene Amplification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
D001426:Bacterial Proteins; C062847:EbgR protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D005696:Galactosidases; D001616:beta-Galactosidase; C062846:evolved beta-galactosidase, E coli