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

Abstract

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.

Nitrate reduction and the nitrogen cycle in archaea

“…The enzyme subunit contains many charged residues that at high temperature arrange networks of ion pairs for proper folding and maintaining the native conformation of the enzyme (Wang et al, 2003). The GDH enzyme of Aeropyrum pernix has also been characterized (Bhuiya et al, 2000;Helianti et al, 2001Helianti et al, , 2002. The enzyme also has a hexameric structure with a molecular mass of about 285 kDa, and is specific for NADP.…”

Temperature Dependence of Kinetic Parameters for Hyperthermophilic Glutamate Dehydrogenase fromAeropyrum pernixK1

“…The Kms for L-glutamate and NADPH of the A. pernix K1 enzyme are high compared to those of GluDH from Pyrococcus furiosus at 509 C, 6) but the Kms are low compared to those at high temperature. 10) Hudson et al has reported the increase in the Km with the temperature for the substrates and coenzymes of the oxidative deamination and the reductive amination of GluDH from isolate AN1.…”

“…[3][4][5] Recently, we puriˆed an NADP-dependent glutamate dehydrogenase (GluDH) from A. pernix K1 and characterized it. 6) In the procedure of characterization, the temperature used for the assay was 509 C which is somewhat lower than that of the optimum growth temperature. This temperature does not re‰ect the physiological conditions of the hyperthermophiles.…”

“…pernix K1 (JCM 9820) was aerobically cultured at 909 C under the same conditions as previously described, 6) and GluDH was puriˆed from the crude extract of the A. pernix K1 cells by the previous methods. 6) The enzyme was assayed spectrophotometrically with a Shimadzu 1600 spectrophotometer equipped with a thermostat (Thermoelectric Cuvette Temperature Controller S140, Shimadzu, Kyoto, Japan) which kept the temperature of reaction mixture in the cuvette between 50-1009 C. The standard reaction mixture for the oxidative deamination was composed of 250 mmol of glycylglycine W NaOH buŠer (pH 8.3), 20 mmol of Lglutamate (pH 8.3), 2.5 mmol of NADP, and the enzyme in aˆnal volume of 2.00 ml. The standard reaction mixture for the reductive amination was composed of 125 mmol of glycylglycine W NaOH buŠer (pH 8.3), 1000 mmol of NH4Cl (pH 8.3, adjusted with NaOH), 30 mmol of sodium 2-oxoglutarate, 0.35 mmol of NADPH, and the enzyme in aˆnal volume of 1.00 ml.…”

“…Protein was measured by the Bradford method as previously described. 6) Temperature dependence of kinetic constants for L-glutamate and NADP in the oxidative deamination, and 2-oxoglutarate, ammonia, and NADPH in the reductive amination of A. pernix K1 GluDH, was investigated. Initial velocity experiments were done at 50 to 909 C as previously described.…”