Construction of aminotransferase chimeras and analysis of their substrate specificity.
Escherichia coli aspartate aminotransferase (AspAT) and E. coli aromatic amino acid aminotransferase (AroAT) have almost identical and high activities toward acidic amino acid substrates. AroAT also has high activity toward aromatic amino acid substrates. The two proteins have 44% amino acid sequence homology. In order to study the mechanism responsible for the different substrate specificities of these aminotransferases, chimeric enzymes of AspAT and AroAT were constructed using homologous recombination in E. coli cells. Five chimeric enzymes were obtained, even though the nucleotide sequence homology between the two parent enzymes was as low as about 50%. The yields of the legitimate chimeric genes were related to the lengths of the homologous region between the two parent genes. Homologous recombination occurred in the region where more than eight nucleotides out of ten were identical. The substrate specificity of the chimeric enzymes suggest that not only the amino acid residues in the active site but also those distant from the active site contribute to the substrate specificity of the parental aminotransferases.
Miyazawa
K
K
Department of Biology, Faculty of Science, Osaka University.
Kawaguchi
S
S
Okamoto
A
A
Kato
R
R
Ogawa
T
T
Kuramitsu
S
S
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D000637:Transaminases; D001219:Aspartate Aminotransferases; C019729:aromatic amino acid aminotransferase