Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications.
NADP-dependent glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95 degrees C for 30 min, and the maximum activity in L-glutamate deamination was obtained around 100 degrees C. The enzyme showed a strict specificity for L-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The Km values for NADP, L-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the 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 1257 bp 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 Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum 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 tree coincided with that of 16S rDNAs obtained from the same organisms.
Bhuiya
M W
MW
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Japan.
Sakuraba
H
H
Kujo
C
C
Nunoura-Kominato
N
N
Kawarabayasi
Y
Y
Kikuchi
H
H
Ohshima
T
T
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019620:Desulfurococcaceae / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase