Preparation of Chimeric Enzymes and Their Properties

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The "homologous recombination" method is an in vivo ligation method that is independent of restriction-endonuclease site. The limit of usefulness of this method was checked. First, a 40-50-mer double stranded oligonucleotide is synthesized. This oligonucleotide includes the sequence around the junction in the final expected product. This synthetic oligonucleotide is then inserted up or down stream from the original DNA fragment. The plasmid is cleaved by a restriction endonuclease within two homologous regions. When E.Coli JC8679 (recBC,sbcA) is transformed with this linearized plasmid, the recombination occurs between the two homologous regions in the cell. We have developed the "homologous ligation" method for overproducing chimeric enzymes and an extremely thermophilic enzyme of aminotransferases.Aminotransferases catalyze the reversible transamination reaction via the ping-pong bi-bi mechanism. Escherichia coli aspartate aminotransferase (AspAT) and aromatic amino acid aminotransfe … More rase (AroAT) have high specificity for both acidic and hydrophobic substrates. This interesting phenomenon was analyzed by site-directed mutagenesis, chimera studies, X-ray crystallography and steady-state and presteady-state kinetic studies.Some chimeric enzymes constructed by homologous recombination in E.coli cells lost their activity for either the acidic or hydrophobic substrate, but retained their activity for the other. These results suggest that aminotransferases have two substrate-binding sites for acidic and hydrophobic substrates and that construction of two substrate-binding pockets might be a general strategy employed by transferases.We investigated the activity of aminotransferases using a series of aliphatic substrates. Enzyme activity was found to increase with an increase in substrate hydrophobicity. For large hydrophobic substrates, the enzyme did not distinguish their shapes. These results indicated that the substrate-binding pocket had a uniform hydrophobic environment and that steric hindrance for the substrate was very weak. Less