Properties and primary structure of the L‐malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus

Abstract

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deduced from the coding gene.The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specifity for NAD' or NADP' and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52"C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism.With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes.The phylogenetic meaning of this relationship is discussed.Archaebacteria differ from eubacteria and eukaryotes in many respects, encouraging studies on the physiological and biochemical bases of their phenotype as well as on their phylogenetic position within the living world.One of the most striking features of many archaebacteria is represented by the ability to grow under extreme conditions, i.e. at high salinity, low pH, or at extremely high temperatures. In order to understand the biochemical background of thermoadaptation, we have been investigating the structure and function of enzymes from extremely thermophilic archaebacteria.Sequence comparisons of glyceraldehyde-3-phosphate dehydrogenases [phosphorylating ~-glyceraldehyde-3-phosphate : NAD(P)+ oxidoreductase (E.C. 1.2.1 . -)I from closely related meso-and thermophilic methanogenic bacteria [l] provided preliminary evidence for the structural prerequisites of thermoadaptation. To analyze the structure/function relationship of thermophilic enzymes from archaebacteria on a broader basis, we included L-malate dehydrogenase in our studies.Like glyceraldehyde-3-phosphate dehydrogenase, L-malate dehydrogenase, occurs in all three urkingdoms. Detailed structural analyzes, however, have only been done with eubacterial and eukaryotic enzymes [2 -101. Thus, structural work on the archaebacterial L-malate dehydrogenase should provide new information, not only for the biochemical Enzymes. L-Malate dehydrogenase (NAD+) (EC 1.1.1.37); L-malate dehydrogenase (NADP') (EC 1.1.1.82); ~-glyceraldehyde-3-phosphate dehydrogenase (NAD') (EC 1.2.1.12); D-glyceraldehyde-3-phosphate dehydrogenase (NAD+/NADP') (EC 1.2.1.-); L-lactate dehydrogenase (EC 1.1.1.27).Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X51714. The novel amino acid sequence data have been deposited with the EMBL sequence data bank.thermoadaptation, but also for the evolutionary differentiation of the enzyme throughout all three urkingdoms.Here we describe some phenotypic properties of L-malate dehydrogenase from the extremely thermop...

Citrate synthase from the thermophilic archaebacterium Thermoplasma acidophilum

“…From the multiple sequence comparisons, it appears that very little similarity with either eukaryotic or eubacterial citrate synthases can be dis-cerned. Equally divergent sequences between archaebacterial and non-archaebacterial proteins have been previously found for glyceraldehyde-3-phosphate dehydrogenase (in archaebacteria sequenced from several methanogens and Pyrococcus woesei) [33] and for malate dehydrogenase (from Methanothermu.r,fervidu,s) [34]. Again, as for citrate synthase, functionally important regions were conserved.…”

Crystal Structures of Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Belonging to a New Family of NAD(P)H-dependent Oxidoreductases

“…2 l of a protein solution at a concentration of 6 mg/ml were mixed with an equal volume of a reservoir solution containing 0.8 M Na/K tartrate, 20% (v/v) glycerol, and 0.1 M Tris-HCl (pH 7.5) and equilibrated against 400 l of a reservoir solution at 293 K. Within 3 days, crystals had grown to dimensions of ϳ0.15ϫ0.15ϫ0.10 mm 3 . Crystals of PsDpkA⅐NADPH and DpkA⅐NADPH⅐P2CA were obtained at 293 K by soaking the unliganded crystals in a reservoir solution containing 40 mM NADPH for 10 min, and 40 mM NADPH and 10 mM P2CA for 30 min, respectively, before data collection.…”

“…However, MDH from the extremely thermophilic methanogen Methanothermus fervidus and LDH from the Gram-negative bacterium Alcaligenes eutrophus were shown to have no similarity in primary sequence to conventional MDH/LDH family proteins (3,4). These two enzymes and their homologs have thus been annotated as new MDH/LDHs different from the conventional MDH/LDH.…”

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.…”