Jiang F, Hellman U, Sroga G E, Bergman B, Mannervik B
Department of Biochemistry, Uppsala University, Sweden.
J Biol Chem. 1995 Sep 29;270(39):22882-9. doi: 10.1074/jbc.270.39.22882.
Glutathione reductase (GR) was purified from the cyanobacterium Anabaena PCC 7120. A 3-kilobase genomic DNA fragment containing the coding sequence for the GR gene (gor) was identified and cloned by polymerase chain reaction based on sequences of selected peptides isolated from proteolyzed GR. The coding sequence encompassing 458 amino acid residues, as well as 360 base pairs of the 5'-flanking region and 430 base pairs of the 3'-flanking region, were determined. Genomic Southern analysis indicates that gor is a single-copy gene. A gor antisense RNA probe hybridized with a 1.4-kilobase transcript, suggesting that the gene is not part of an operon including additional genes. The deduced GR amino acid sequence shows 41 to 48% identity with those of human, Escherichia coli, Pseudomonas aeruginosa, pea, and Arabidopsis thaliana GR. The coding sequence of GR was overexpressed in a GR-deficient E. coli strain, SG5, and the recombinant protein was purified. Anabaena GR is NADPH-linked, but a Lys residue replaces an Arg residue involved in NADPH binding in GR from other species. In addition, Anabaena GR carries the GXGXXG "fingerprint" motif which otherwise characterizes NAD(H)-dependent enzymes. These differences may contribute to the lack of affinity for 2',5'-ADP-Sepharose 4B of Anabaena GR. Three E. coli-type promoter sequences and a BifA/NtcA binding motif were found upstream of the open reading frame. The middle and the proximal promoters were shown to be active. However, the use of the middle promoter was dependent on the nitrogen source in the culture medium. Both GR activity and GR protein concentration increased in ammonium grown cultures in which both the middle and proximal promoters were used for transcriptional initiation. The BifA/NtcA-binding site overlaps the middle promoter sequence and may thus be involved in regulation of differential transcription.
谷胱甘肽还原酶（GR）是从蓝藻鱼腥藻PCC 7120中纯化得到的。基于从蛋白酶解的GR中分离出的选定肽段的序列，通过聚合酶链反应鉴定并克隆了一个包含GR基因（gor）编码序列的3千碱基基因组DNA片段。确定了包含458个氨基酸残基的编码序列，以及5'侧翼区的360个碱基对和3'侧翼区的430个碱基对。基因组Southern分析表明gor是单拷贝基因。一个gor反义RNA探针与一个1.4千碱基的转录本杂交，表明该基因不是包括其他基因的操纵子的一部分。推导的GR氨基酸序列与人类、大肠杆菌、铜绿假单胞菌、豌豆和拟南芥GR的氨基酸序列具有41%至48%的同一性。GR的编码序列在GR缺陷型大肠杆菌菌株SG5中过表达，并纯化了重组蛋白。鱼腥藻GR与NADPH相连，但一个赖氨酸残基取代了其他物种GR中参与NADPH结合的精氨酸残基。此外，鱼腥藻GR带有GXGXXG“指纹”基序，否则该基序是NAD（H）依赖性酶的特征。这些差异可能导致鱼腥藻GR对2',5'-ADP-琼脂糖4B缺乏亲和力。在开放阅读框上游发现了三个大肠杆菌型启动子序列和一个BifA/NtcA结合基序。中间和近端启动子显示有活性。然而，中间启动子的使用取决于培养基中的氮源。在铵生长的培养物中，GR活性和GR蛋白浓度都增加，在这种培养物中，中间和近端启动子都用于转录起始。BifA/NtcA结合位点与中间启动子序列重叠，因此可能参与差异转录的调控。
