The glutamate dehydrogenase gene of Clotridium symbiosum
The glutamate dehydrogenase gene of Clotridium symbiosum
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共生梭菌的谷氨酸脱氢酶基因
DOI：
10.1111/j.1432-1033.1992.tb16912.x
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发表时间：
1992
期刊：
影响因子：
5.4
通讯作者：
J. Guest
中科院分区：
文献类型：
--
作者：
J. Teller;Rachel J. Smith;M. McPherson;P. Engel;J. Guest
共生梭菌（Clostridium symbiosum）中NAD +依赖性谷氨酸脱氢酶（glutamate dehydrogenase， GDH）的编码基因无法通过标准技术得到，因此采用聚合酶链反应（polymerase chain reaction， PCR）克隆该基因。测定了gdh基因的核苷酸序列，在大肠杆菌中通过可控tac启动子过表达，使活性梭菌gdh占细胞总蛋白的20%。重组质粒补充了大肠杆菌谷氨酸缺乏症的营养损伤。编码区（G + C = 52%）与侧翼序列（G + C = 30%和37%）的核苷酸组成差异显著。该结构基因编码了一个由450个氨基酸残基和相对分子质量（M,） 49295组成的多肽，对应于六聚体酶的一个亚基。dna来源的氨基酸序列与梭状菌酶的色氨酸和溴化氰肽的部分序列一致。n端氨基酸序列与纯化蛋白相匹配，表明起始蛋氨酸在翻译后被去除，就像在天然宿主中一样。它的氨基酸序列与其他细菌的GDHs相似，尽管它在nadf结合区域有一个Gly-Xaa-Gly-XaaXaa-Ala基序，这是更典型的nadf依赖酶。序列数据现在允许对酶的x射线晶体结构进行详细的解释，梭状芽孢杆菌基因的克隆和表达将促进定点诱变。共生梭菌的NAD +依赖性谷氨酸脱氢酶（GDH）催化谷氨酸发酵羟戊二酸途径的第一步11,21。这种厌氧菌是GDH的丰富来源，在谷氨酸生长过程中，GDH约占细胞总蛋白的15%。该酶已被纯化和广泛表征（3 - 51），代表约75%初级结构的肽已被测序161。此外，C. symbiosum GDH已结晶成适合高分辨率x射线分析的形态，其结构已解算到0.196 nm [7-lo]。gdh基因的克隆及其核苷酸序列的确定对于推断该酶的完整氨基酸序列、允许对分子结构的完整解释以及设计定点诱变实验具有重要意义。共生菌gdh基因的常规克隆方法
The gene encoding the NAD +-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M,) 49 295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-XaaXaa-Ala motif in the NADf-binding domain, which is more typical of the NADPf-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate sitedirected mutagenesis. The NAD +-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum catalyses the first step in the hydroxyglutarate pathway of glutamate fermentation 11, 21. This anaerobe is an abundant source of GDH, which amounts to approximately 15% of the total cell protein during growth with glutamate. The enzyme has been purified and extensively characterized (3 - 51 and peptides representing about 75% of the primary structure have been sequenced 161. In addition, C. symbiosum GDH has been crystallized in a form suitable for high-resolution X-ray analysis and the structure has been solved to 0.196 nm [7-lo]. The cloning of the gdh gene and the determination of its nucleotide sequence are important for deducing the complete amino acid sequence of the enzyme, for permitting a full interpretation of the molecular structure and for designing site-directed mutagenesis experiments. Conventional approaches for cloning the gdh gene of C. symbiosum
DOI：
10.1073/pnas.84.14.4767
发表时间：
1987-07-01
影响因子：
11.1
作者：
TABOR, S;RICHARDSON, CC
通讯作者：
RICHARDSON, CC
全长小鼠脑谷氨酸脱氢酶 cDNA 的分子克隆、结构和表达分析。
DOI：
10.1016/0167-4781(91)90017-g
发表时间：
1991
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Tzimagiorgis,G;Moschonas,NK
通讯作者：
Moschonas,NK