Fusion of trpB and trpA of Escherichia coli yields a partially active tryptophan synthetase polypeptide.
The separate alpha and beta polypeptides of the tryptophan synthetase of bacteria are represented in fungi by a fusion polypeptide in which the first third is homologous to bacterial alpha chains and the remainder is homologous to bacterial beta chains. In the yeast polypeptide, a short nonhomologous "connector" joins the two homologous segments. The chromosomal order of all bacterial genes that specify tryptophan synthetase beta and alpha chains, respectively, is trpB-trpA. Fusion of these genes in their present arrangement would result in the synthesis of a polypeptide with a segmental order, N-beta-alpha-C, opposite that observed in fungi. To investigate possible explanations for the apparent transposition that occurred in the evolution of the fungal gene we have made two fusions of trpB and trpA of Escherichia coli in their natural orientation. We find that the fusion proteins are synthesized but both are less active catalytically than the wild type bacterial protein. In addition, the fusion proteins associate abnormally, they are activated only slightly by wild type alpha or beta 2, and they are less sensitive than the wild type protein to inhibition by antibodies to alpha or beta 2. The fusion proteins have normal substrate affinities. Our findings suggest that the altered structures of the fusion proteins affect catalytic ability and the locations of the alpha and/or beta chain combining sites. This structural distortion may have prevented the natural selection of direct gene fusions during the course of the fungal gene's evolution.
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014367:Tryptophan Synthase / Q000235:genetics*
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014367:Tryptophan Synthase