Creation and design of novel enzymes by analysis and modification of nucleotide sequence in DNA and its application

大肠杆菌B的谷胱甘肽合成酶（GSH-II）在5'-三磷酸腺苷（ATP）存在下催化< γ >-谷氨酰半胱氨酸和甘氨酸合成谷胱甘肽。为了分析该酶的功能和结构之间的关系，我们首先寻找与GSH-II具有同源氨基酸序列的蛋白质，发现二羟叶酸还原酶（dihydroxyfolate reductase， FDR）在氨基酸序列上具有约70%的同源区域。利用两种酶（GSH-II和FDR）的抑制剂和/或底物进行酶分析表明，同源区域代表两种酶的催化结构域。其次，为了了解半胱氨酸残基对GSH-II活性和/或稳定性的贡献，通过位点定向诱变将GSH-II亚基中半胱氨酸的4个密码子分别改变为丙氨酸的密码子。虽然这两种替代都不影响活性，但超过2个半胱氨酸残基的替代导致GSH-II活性显著降低。因此，GSH-II亚基中的4个半胱氨酸残基被认为在GSH-II活性的充分表达中起重要作用。< γ >-谷氨酰半胱氨酸合成酶（GSH-I）催化谷胱甘肽合成的第一步，在ATP存在下由谷氨酸和半胱氨酸生成< γ >-谷氨酰半胱氨酸。gsh - 1的基因有一个不寻常的起始密码子TTG。通过定点诱变，将TTG密码子替换为一般起始密码子ATG。结果，该基因的转录效率提高了50%以上。

Glutathione synthetase (GSH-II) of Eschrichia coli B catalyzes the synthesis of glutathione from <gamma>-glutamylcysteine and glycine in the Presence of adenosine 5'-triphosphate (ATP).To analyze the relation between function and structure of the enzyme,we first searched for proteins with a homologous amino acid sequence with that of GSH-II and found that an enzyme,dihydroxyfolate reductase(FDR)has a region approximately 70% homologous in amino acid sequence.The enzymatic analyses using inhibitors and/or substrates of the two enzymes (GSH-II and FDR) indicated that the homologous region represented the catalytic domain on the two enzymes.Secondly,to know the contribution of cysteine residues on activity and/or stability of the GSH-II,the 4 codons for cysteine in the GSH-II subunit were severally changed to the codons for alanine by site-directed mutagenesis.Although either replacement did not affect the activity,the replacements of more than 2 cysteine residues resulted in the significant decrease in the GSH-II activity. The 4 cysteine residues in the GSH-II subunit were,therefore,thought to play an important role in the full expression of the GSH-II activity.<gamma>-Glutamylcysteine synthetase(GSH-I)catalyzes the first step of glutathione synthesis and produces <gamma>-glutamylcysteine from glutamate and cysteine in the presence of ATP.The gene for the GSH-I has an unusual initiation codon TTG.The codon (TTG)was replaced with general initition codon ATG by site-directed mutagenesis.As a result, the transcriptional efficiency of the gene was increased more than 50%.