Kort R, Liebl W, Labedan B, Forterre P, Eggen R I, de Vos W M
Department of Microbiology, Wageningen Agricultural University, The Netherlands.
Extremophiles. 1997 Feb;1(1):52-60. doi: 10.1007/s007920050014.
The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90 degrees C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45,852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH2-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2-13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75 degrees C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (Vmax of 172 U/mg protein), but also utilizes NADPH (Vmax of 12 U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota.
嗜热栖热菌（Thermotoga maritima）能在高达90摄氏度的环境中生长，它含有一种L-谷氨酸脱氢酶（GDH）。在嗜热栖热菌的粗提取物中可检测到这种酶的活性，其活性似乎与一种47 kDa的蛋白质相关，该蛋白质能与抗嗜热古菌沃氏嗜热栖热菌（Pyrococcus woesei）纯化GDH的抗体发生交叉反应。通过在谷氨酸营养缺陷型大肠杆菌菌株中进行互补克隆出了单拷贝的嗜热栖热菌gdh基因。gdh基因的核苷酸序列预测出一个由416个残基组成的蛋白质，计算分子量为45,852。将gdh基因插入表达载体并在大肠杆菌中表达出活性酶。嗜热栖热菌GDH被纯化至同质。纯化酶的NH2末端序列为PEKSLYEMAVEQ，与从gdh基因推导的肽序列的第2至13位相同。纯化后的天然酶大小为265 kDa，亚基大小为47 kDa，表明GDH是一种同六聚体。该酶的最大活性在75摄氏度时测得，合成代谢和分解代谢反应的最适pH分别为8.3和8.8。发现该酶在80摄氏度时非常稳定，但在更高温度下似乎会迅速失去活性。嗜热栖热菌GDH对NADH的活性速率最高（Vmax为172 U/mg蛋白质），但也利用NADPH（Vmax为12 U/mg蛋白质）。序列比较表明，嗜热栖热菌GDH是六聚体GDH家族II的成员，该家族包括迄今从嗜热菌中分离出的所有GDH。值得注意的是，系统发育分析将所有这些嗜热GDH定位在GDH家族II树的中间位置，细菌来源的嗜热栖热菌GDH位于嗜盐和嗜热广古菌的GDH之间。
