Characterization of two d‐glyceraldehyde‐3‐phosphate dehydrogenases from the extremely thermophilic archaebacterium Thermoproteus tenax

Abstract

Thermoproteus tenax possesses two different glyceraldehyde-3-phosphate dehydrogenases, one specific for NADP' and the other for NAD' . NADP(H) inhibits the NAD+-specific enzyme competetively with respect to NAD + whereas NAD(H) virtually does not interact with the NADP+-specific enzyme. Both enzymes represent homomeric tetramers with subunit molecular masses of 39 kDa (NADP+-specific enzyme) and 49 kDa (NAD+-specific enzyme), respectively.The NADP+-specific enzyme shows significant homology to the known glyceraldehyde-3-phosphate dehydrogenases from eubacteria and eukaryotes as indicated by partial sequencing.The enzymes are thermostable, the NADP+-specific enzyme with a half-life of 35 min at lOO"C, the NAD+-specific enzyme with a half-line of 220min at lOO"C, depending on the protein concentration. Both enzymes show conformational and functional changes at 60 -70°C.The existence of the extremely thermophilic sulfur-dependent archaebacteria, with growth optima near 100°C and above [l -41, raises questions concerning the thermo-adaptations of their enzyme proteins.Here we describe two ~-glyceraldehyde-3-phosphate dehydrogenases from the sulfur-dependent archaebacterium Thermoproteus tenax (optimal growth temperature 88 "C; upper growth limit 96°C [4]), which differ in their catalytic and molecular properties, and characterize their thermophilic behavior regarding activity and stability. MATERIALS AND METHODS Chemicals and reagentsThe origin of most of the chemicals used and the preparation methods for the enzyme substrates are given in a previous publication [5].Freund's complete adjuvant was obtained from Difco laboratories (Detroit, USA), [U-'4C]sucrose and 3 H 2 0 were from Amersham-Buchler (Braunschweig, FRG), NADPSepharose was from Pharmacia (Uppsala, Sweden).Methods for protein determination, analytical gel chromatography, preparation of polyacrylamide/dodecyl sulfate slab gel electrophoresis, amino acid analysis, tryptic digestion, peptide fractionation, sequencing using 4-dimethylaminoazobenzene-4'-isothiocyanate, and comparisons of peptide sequences and calculation of the activation parameters were described previously [5]. steel fermenters (Bioengineering, Wald, Switzerland) (heterotrophic growth). Cultures were gassed with C 0 2 and, for autotrophic growth, with a mixture of 20% H2 and 80% C 0 2 at 88 "C. The basic medium was as described by Brock et al. [6]. For heterotrophic growth 1% glucose and 0.04% yeast extract were added. Cells were harvested at the late-exponential phase. After cooling the cultures using a heat exchanger and passing through a folded filter (520b 1/2 Schleicher & Schiill, Dassel, FRG) for removing the sulfur, the cells were centrifuged, frozen and stored in liquid nitrogen. Organisms and growth conditions Enzyme assayOxidation of glyceraldehyde 3-phosphate. A previously described procedure [5] was generally followed. The standard assay mixture (pH 7.0 at 70°C) contained 100 mM Tris/HCl, 20 mM 2-mercaptoethanol, 150 mM potassium arsenate, 4 mM ~-glyceraldehyde-3-phosphate and ...

Characterization of a halophilic glyceraldehyde-3-phosphate dehydrogenase from the archaebacterium Haloarcula vallismortis.

“…From the co-electrophoresis patterns of the halobacterial and mammalian GAPDH it is evident that hGAPDH is the larger of the two, though the difference is not great. But the subunit size of hGAPDH falls within the range 37 to 49 kDa, as also reported for its counterparts from the other two archaebacterial groups (17,18,24). However it would be better to use alternate methods to determine hGAPDH molecular weight.…”

“…Since NAD+ does not stabilize the enzyme any further, it appeared that conditions that favour the hydrophobic interactions are responsible for the thermostability of the hGAPDH. The enhanced stabilization at higher temperatures in the presence of phosphate also occurs in GAPDHs from thermophilic and methanogenic bacteria (17,24). Many members of the Methanobacteriaceae have elevated levels of KCl but it is not known whether M. fervidus also has equally high internal K } concentration (17).…”

“…Recently much work has been published on the GAPDHs from thermophilic (24,25) and methanogenic archaebacteria (17)(18)(19)(20). But there is hardly any information available on GAPDH from halobacteria, the group placed in the third kingdom of life, the archaebacteria (21).…”

Evidence for a chimeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3-phosphate dehydrogenase genes.

“…The lower cladogram depicts schematically the same relationships observed for GAPDH. The anomalous evolutionary pattern for GAPDH was first noted through the cloning (35,44,45) and phylogenetic analysis (34) of the archaebacterial genes. (B) Schematic depiction of lateral endosymbiotic transfer which could account for various aspects of the data (see text).…”

Functional expression of d‐glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima in Escherichia coli

“…In this context, ~-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appears to be a suitable model system to investigate the correlation of structure, function and energetics over a wide temperature range. The enzyme from various mesophilic and thermophilic sources has been studied in detail, including numerous sequence determinations and high-resolution X-ray analyses of three homologs (Buehner et al, 44 1973;Harris and Waters, 1976;Biesecker et al, 1977;Hensel et al, 1987;Skarzynski et al, 1987;Fabry et al, 1988;Schultes et al, 1990).…”