Purification and characterization of d‐glyceraldehyde‐3‐phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus

Abstract

The ~-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the ~-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD' and NADP' and is not inhlbited by pentalenolactone. The enzyme is intrinsically stable up to 75°C. It is stabilized by the coenzyme NADP' and at high ionic strength up to about 90°C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52°C.Despite numerous studies on enzymes from thermophilic organisms mainly isolated from eubacterial and eukaryotic sources [ 11 the structure principles which enable these enzymes to be stable and active at high temperature remain unclear. Detailed structural comparisons between mesophilic and thermophilic enzyme homologues, especially of D-glyCeraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase and ferredoxin [2-51 did not lead to a consensus of structural details responsible for stability and activity at high temperature. In some cases, however, the phylogenetic distances between the mesophilic and thermophilic organisms used in these comparisons may be too large for detecting thermophily-specific structures. Also, the detailed investigations on different temperature-sensitive mutants of tryptophan synthetase, lysozyme and A repressor [6 -81 deal only with the thermostability and thus do not consider the whole spectrum of biochemical adaptations to high temperature.The discovery of extremely thermophilic archaebacteria gives rise to the hope that the enzyme proteins of these organisms could show the structural requisites for the thermophilic behaviour more clearly than the proteins of the rather moderately thermophilic eubacteria and eukaryotes investigated up to now.Thermophilic archaebacteria were found among the methanogens and in the so-called sulfur-dependent group. Within the methanogens closely related mesophilic and thermophilic organisms provide an ideal basis for comparisons between mesophilic and thermophilic enzyme proteins, the sulfur-dependent archaebacteria comprise exclusively thermophilic organisms with Pyrodictium occultum Enzymes. ~-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12); ~-glyceraldehyde-3-phosphate dehydrogenase (NADP') (phosphorylating) (EC 1.2.1.13); trypsin (EC 3.4.21.4); L-lactate dehydrogenase (EC 1.1.1.27); D-lactate dehydrogenase (EC 1.1.1.28); cytochrome c reductase (EC 1.9.99.1); catalase (EC 1.11.1.6).coccus furiosus [lo] and Pyrococcus woesei [ l l ] as the most thermophilic organisms known as yet.For analyzing the structure and function of thermophilic archaebacterial enzymes we focus on the ~-glyceraldehyde-3-phosphate dehydrogenase because this enzyme, at least the enzyme homologues from mesophilic and thermophilic eubacteria...

Dimeric 3‐Phosphoglycerate Kinases from Hyperthermophilic Archaea

“…Despite some scattering of the data, mainly due to the instability of the substrates glycerate-3-P and NADPH at higher temperatures, a rather linear, dependence of 1n(Vn,J on 1/T could be deduced over the temperature range tested. In contrast, non-linear Arrhenius plots have been described for several enzymes from thermophilic Archaea [15,41,421 and Bacteria [43, 441 and interpreted as indicative of temperature-induced conformational changes. Additional investigations (e.g.…”

“…Activities of 3-phosphoglycerate kinase from f? woesei and M. ,fervidus were determined photometrically at 366 nm and 70°C with equipment described earlier [15]. Standard assay conditions (total volume 1 ml) for the F1 cvoesei enzyme were 100 mM Hepes, 0.6 M KCI, 10 mM MgCI,, 10.5 mM ATP, 13.5 mM glycerate-3-P, 0.2 mM NADPH, and 2 U of GraP-DH from f?…”

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

“…The origin of most of the chemicals used and the preparation methods for the enzyme substrates are given in a previous publication [5].…”

“…The thermolabile D-glyceraldehyde 3-phosphate (100 mM stock solution) was added immediately before the reaction was started by the enzyme. 1 unit of enzyme activity represents the amount of enzyme which catalyzes the formation of 1 pmol NADH or NADPH/min at 70 "C. The temperature-dependent decrease of the molar decadic absorption coefficient of NADPH was determined as recently described for NADH [5] and shown in Fig. 1.…”

“…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).…”