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The gene encoding streptococcal NAD^+-glycohydrolase : datermination of primary structure and analysis of enzymatic active sites

编码链球菌NADβ-糖水解酶的基因：一级结构的测定和酶活性位点的分析

基本信息

批准号：
08670303
负责人：
KARASAWA Tadahiro
金额：
$ 1.41万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670303/
关键词：
Streptococcus pyogenes NAD cyclic ADP-ribose cloning サイクリックADPリボースクローニング

项目摘要

1. Cloning of a DNA fragment corresponding to the N-terminus amino acid sequence of streptoccal NAD^+-glycohydrolaseAn N-terminus amino acid sequence of streptococcal NAD^+-glycohydrolase was determined by using the purified enzyme. To amplify a DNA fragment which corresponded to the N-terminus amino acid sequence, PCR-primers were designed and PCR was done. A DNA fragment which had the expected size (120 bp) was amplified. The nucleotide and the deduced amino acid sequences of this fragment revealed that this fragment was a part of the gend encoding NAD^+-glycohydrolase.2. Primary structure of the gene encoding NAD^+-glycohydrolase and construction of its expression systemSouthern blot analysis was done using the 120-bp DNA fragment. It was shown that the gene encoding NAD^+-glycohydrolase was contained as a single copy in chromosome. Plasmid libraries of chromosomal DNA from Streptococcus pyogenes C2556 were constructed. Single specific-PCR was preformed using primers designed from sequences of the plasmid and of the 120-bp DNA fragment. The nucleotide sequence of the amplicon was determined. The open reading frame was ca.1400 bp. Calculated molecular weight and pI were ca.50,000 and 8.5, respectively. These properties were consistent with those of the purified enzyme. Full length of the gene encoding NAD^+-glycohydrolase was cloned and expressed in Escherichia coli. Cell extracts showed not only NAD^+-glycohydrolase activity bu also cyclic ADP-ribose-synthesis and -hydrolysis activities. Enzymatic active portions are currently under investigation.

1.与链球菌NAD^+-糖水解酶N-末端氨基酸序列相对应的DNA片段的克隆使用纯化的酶测定链球菌NAD^+-糖水解酶的N-末端氨基酸序列。为了扩增对应于N-末端氨基酸序列的DNA片段，设计PCR引物并进行PCR。扩增了具有预期大小（120 bp）的DNA片段。该片段的核苷酸序列和推导的氨基酸序列表明，该片段是NAD^+-糖苷水解酶基因的一部分. NAD^+-葡萄糖水解酶基因的一级结构及其表达系统的构建用120 bp的DNA片段进行Southern杂交分析。结果表明，编码NAD^+-糖水解酶的基因以单拷贝形式存在于染色体中。构建了化脓性链球菌C2556染色体DNA的质粒文库。使用从质粒和120-bp DNA片段的序列设计的引物进行单特异性PCR。测定扩增子的核苷酸序列。开放阅读框约为1400 bp。计算的分子量和pI分别为约50，000和8.5。这些性质与纯化的酶的性质一致。在大肠杆菌中克隆并表达了NAD^+-糖水解酶基因的全长。细胞提取物不仅具有NAD^+-糖水解酶活性，而且还具有环ADP-核糖-合成和-水解活性。酶活性部分目前正在研究中。