Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications.
The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90 degrees C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45,852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH2-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2-13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75 degrees C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (Vmax of 172 U/mg protein), but also utilizes NADPH (Vmax of 12 U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota.
Kort
R
R
Department of Microbiology, Wageningen Agricultural University, The Netherlands.
Liebl
W
W
Labedan
B
B
Forterre
P
P
Eggen
R I
RI
de Vos
W M
WM
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D005969:Glutamate Dehydrogenase