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). Activit y of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which crossreacted wit h 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. Th e nucleotide sequence of the gdh gene predicts a 416-residue protein w ith a calculated molecular weight of 45852. The gdh gene was inserted in an expression vector coli as an active enzyme. The purified to homo geneity, The NH2-terminal sequence of the purified enzyme was PEKSLYEM AVEQ, which is identical to positions 2-13 of the peptide sequence der ived from the gdh gene, The purified native enzyme has a size of 265 k Da and a subunit size of 47 kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75 degrees C and the p H optima are 8.3 and 8.8 for the anabolic and catabolic reaction, resp ectively. The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures, The T. marit ima GDH shows the highest rate of activity with NADH (V-max of 172U/mg protein), but also utilizes NADPH (V-max of 12U/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 th ese hyperthermophilic GDHs in the middle of the GDH family II tree, wi th the bacterial T. maritima GDH located between that of halophilic an d thermophilic euryarchaeota.