Preparation of Chimeric Enzymes and Their Properties

“同源重组”方法是一种不依赖于限制性内切酶位点的体内连接方法。检验了该方法的适用范围。首先，合成一个40-50聚体的双链寡核苷酸。该寡核苷酸包括最终预期产物中连接处附近的序列。然后将这种合成的寡核苷酸插入到原始DNA片段的上游或下游。该质粒在两个同源区域内被限制性内切酶切割。用线性化后的重组质粒转化E.Coli JC8679(recBC，SBCA)，重组发生在细胞内两个同源区域之间。我们开发了一种高效生产嵌合酶的“同源连接”方法和一种极端嗜热性的氨基转移酶，氨基转移酶通过乒乓bi-bi机制催化可逆转氨化反应。大肠杆菌天冬氨酸氨基转移酶和芳香族氨基酸氨基转移酶…更多的Rase(AroAT)对酸性和疏水性底物都具有高度的特异性。通过定点突变、嵌合体研究、X射线结晶学以及稳态和稳态前动力学研究对这一有趣的现象进行了分析。在大肠杆菌中，通过同源重组构建的一些嵌合酶失去了对酸性或疏水性底物的活性，但对另一种底物保持了活性。这些结果表明，转氨酶对酸性底物和疏水性底物有两个底物结合位点，构建两个底物结合口袋可能是转氨酶的一般策略。酶活性随底物疏水性的增加而增加。对于大的疏水底物，酶不能区分它们的形状。这些结果表明，底物结合袋具有均匀的疏水环境，对底物的空间位阻很小。较少

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