Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1:: enzymatic characterization, identification of the encoding gene, and phylogenetic implications
Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1:: enzymatic characterization, identification of the encoding gene, and phylogenetic implications
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DOI：
10.1007/s007920070002
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发表时间：
2000-12-01
期刊：
影响因子：
2.9
通讯作者：
Ohshima, T
中科院分区：
文献类型：
--
作者：
Bhuiya, MW;Sakuraba, H;Ohshima, T
来自需氧超高温古菌 Aeropyrum pernix K1 (JCM 99820) 的 NAPD 依赖性谷氨酸脱氢酶（L-谷氨酸：NAPD 氧化还原酶，脱氨，EC 1.4.1.4）被纯化至均质用于表征。该酶在95℃加热30min后仍保持其全部活性，并且L-谷氨酸脱氨基作用在100℃左右获得最大活性。该酶对氧化脱氨中的 L-谷氨酸和 NADP 以及还原氨化中的 2-酮戊二酸和 NADPH 显示出严格的特异性。 NADP、L-谷氨酸、N-末端氨基酸序列的K-m值，编码基因在A.pernix K1基因组中被鉴定，在大肠杆菌中克隆和表达。核苷酸序列分析显示，有一个 1257bp 的开放阅读框，以小 TTG 密码子开头，编码 418 个氨基酸、分子量为 46170 的蛋白质。系统发育分析显示，A. pernix K1 的谷氨酸脱氢酶与 Crenarchaeota 中需氧 Pryobaculum islandicum 的谷氨酸脱氢酶聚类在一起，并且与来自 Thermococcales 中 Thermococcales 的另一酶聚类分离。广古菌门。 A. Pernix K1、S. solfataricus、S. shibatae 和 Pb 的酶的分支模式。 islandicum 的系统发育三期与从同一生物体获得的 16S rDNA 一致。
NAPD-dependent glutamate dehydrogenase (L-glutamate: NAPD oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 99820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95 degreesC for 30min, and the maximum activity in L-glutamate deamination was obtained around 100 degreesC. The enzyme showed a strict specificity for L-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The K-m values for NADP, L-glutamate, N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46170. Phylogenetic analysis revealed that the glutamate dehydrogenase from, A. pernix K1 clustered with those from aerobic Pryobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. Pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic three coincided with that of 16S rDNAs obtained from the same organisms.