Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs
Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs
复制标题
超嗜热古菌火球菌属谷氨酸脱氢酶 (GDH) 的序列分析。
DOI：
10.1007/s004380050655
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
T. Imanaka
中科院分区：
文献类型：
--
作者：
Raja Noor Zaliha Abd. Rahman;S. Fujiwara;M. Takagi;T. Imanaka
摘要克隆了嗜热古菌Pyrococcus sp.KOD1谷氨酸脱氢酶（GDH）的gdhA基因，并测定了其序列。对包括KOD 1-GDH在内的25个GDH序列进行了系统发育分析，并区分了两个蛋白质家族。KOD 1-GDH是六聚体GDH家族II的新成员。在大肠杆菌中表达并纯化了重组KOD 1-GDH。将其酶特性与天然KOD 1-GDH的酶特性进行比较。这两种酶的分子量为47 - 300 Da，并被证明是功能的六聚体形式（284 kDa）。天然KOD 1-GDH和重组GDH的N-末端氨基酸序列分别为VEIDPFEMAV和MVEIDPFEMA，表明天然KOD 1-GDH不保留N-末端的初始甲硫氨酸。重组GDH显示出与天然GDH相似的酶特性，除了较低水平的热稳定性，在100° C下半衰期为2小时，而从KOD 1纯化的天然酶为4小时。 动力学研究表明，该反应偏向于谷氨酸盐的产生。KOD 1-GDH与大多数真核GDH一样利用辅酶NADH和NADPH。
Abstract The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47 300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100° C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.
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