Protein Engineering of Aspartate Aminotransferase

转氨酶催化氨基酸和α酮酸之间的转氨化。大肠杆菌天冬氨酸转氨酶（AspAT）对酸性底物具有高特异性，而大肠杆菌芳香氨基酸转氨酶（AroAT）对酸性和疏水底物均具有高特异性。两种蛋白的氨基酸序列同源性仅为40%，核苷酸序列同源性为50%。大肠杆菌AspAT的x射线晶体学研究已经揭示了酸性底物的结合位点，但疏水底物的结合位点仍然未知。AspAT或AroAT的定点诱变未能揭示疏水口袋。因此，为了寻找疏水口袋，我们构建了AspAT-AroAT嵌合蛋白。在核苷酸同源性低至50%的情况下，获得了嵌合蛋白。嵌合基因的产率与两个转氨酶之间的同源区长度有关。重组发生在10个核苷酸中有8个相同的区域。提示离活性位点较远的氨基酸残基对转氨酶的底物特异性也有影响。

Aminotransferases catalyze transamination between amino acids and alphaketo acids. Escherichia coli aspartate aminotransferase (AspAT) has high specificity for acidic substrates, whereas E. coli aromatic amino acid aminotransferase (AroAT) has high specificity for both acidic and hydrophobic substrates. The two proteins have only 40% amino acid sequence homology, and 50% nucleotide sequence homology. X-ray crystallographic studies of E. coli AspAT have revealed the binding site for the acidic substrate, but that for the hydrophobic substrate is still unknown. Site-directed mutagenesis of AspAT or AroAT has failed to reveal the hydrophobic pocket. Therefore, in order to search for the hydrophobic pocket, we constructed AspAT-AroAT chimeric proteins.The chimeric proteins were obtained, even though the homology of the nucleotides was as low as 50%. The yield of chimeric gene was related to the length of homologous region between the two aminotransferase.The recombination occurred in the region where eight nucleotides out of ten were identical. It is suggested that the amino acid residues distant from active site contribute also contribute to the substrate specificity of aminotransferase.