Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: Molecular characterization and phylogenetic implications

Remco Kort, Wolfgang Liebl, Bernard Labedan, Patrick Forterre, Rik I.L. Eggen, Willem M. De Vos

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90°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 crossreacted 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 45852. 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 47 kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75°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°C. but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (Vmax of 172U/mg protein), but also utilizes NADPH (Vmax 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 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.

Original languageEnglish
Pages (from-to)52-60
Number of pages9
JournalExtremophiles
Volume1
Issue number1
DOIs
StatePublished - 1997
Externally publishedYes

Keywords

  • Gene cloning and expression
  • Glutamate dehydrogenase
  • Phylogenetic analysis
  • Sequence analysis
  • Thermostability

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