Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1:: enzymatic characterization, identification of the encoding gene, and phylogenetic implications

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.