Archaeon Thermoplasma Acidophilum Research Articles

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum.

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.

Hydration and Molecular Motions in Synthetic Phytanyl-Chained Glycolipid Vesicle Membranes

Proton permeation rates across membranes of a synthetic branch-chained glycolipid, 1,3-di- O-phytanyl-2- O-( β- d-maltotriosyl)glycerol (Mal 3(Phyt) 2) as well as a branch-chained phospholipid, diphytanoylphosphatidylcholine (DPhPC) were lower than those of straight-chained lipids such as egg yolk phosphatidylcholine (EPC) by a factor of ∼4 at pH 7.0 and 25°C. To examine whether degrees of water penetration and molecular motions in Mal 3(Phyt) 2 membranes can account for the lower permeability, nanosecond time-resolved fluorescence spectroscopy was applied to various membranes of branch-chained lipids (Mal 3(Phyt) 2, DPhPC, and a tetraether lipid from an extremely thermoacidophilic archaeon Thermoplasma acidophilum), as well as straight-chained lipids (EPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and digalactosyldiacylglycerol (DGDG)) using several fluorescent lipids. Degrees of hydration of glycolipids, Mal 3(Phyt) 2, and DGDG were lower than those of phospholipids, EPC, POPC, and DPhPC at the membrane-water interfaces. DPhPC showed the highest hydration among the lipids examined. Meanwhile, rotational and lateral diffusive motions of the fluorescent phospholipid in branch-chained lipid membranes were more restricted than those in straight-chained ones. The results suggest that the restricted motion of chain segments rather than the lower hydration accounts for the lower proton permeability of branch-chained lipid membranes.

Tricorn protease in bacteria: characterization of the enzyme from Streptomyces coelicolor.

Tricorn protease is believed to act downstream of the proteasome, or of other ATP-dependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricorn-like genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRI-ScC77 and TRI-ScE87) are very similar in their PDZ and TSP domains, but rather divergent in their beta-propeller domains. We have expressed one of them, TRI-ScC77, in E. coil and characterized the recombinant protein structurally and functionally. TRI-ScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coil and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. The fact that Tricorn-like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.

Cloning and Characterization of ftsZ and pyrF from the Archaeon Thermoplasma acidophilum

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5′-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif.

Why Does Threonine, and Not Serine, Function as the Active Site Nucleophile in Proteasomes?

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.

Monomolecular organization of the main tetraether lipid from Thermoplasma acidophilum at the water–air interface

The monomolecular organization of the main tetraether phospholipid from the archaeon Thermoplasma acidophilum was studied by means of a Langmuir film balance integrated into a fluorescence microscope. After transfer to solid surfaces at different pressures the films were further investigated by ellipsometry, small angle X-ray scattering and atomic force microscopy. In order to complete former results about the main tetraether phospholipid of T. acidophilum [Strobl, C., Six, L., Heckmann, K., Henkel, B., Ring, K., 1985. Z. Naturforsch. 40c, 219–222], the thickness and the two-dimensional organization of the monomolecular films were investigated. Two mean heights values were determined, one of 1.5–1.8 nm and another one of 4–5 nm, indicative for two different molecular arrangements. The former one is interpreted as a ‘horseshoe’ organization with two polar endings in the aqueous subphase, whereas the latter appears to represent the upright population of molecules with one polar end in the subphase and the other one in the air. In freshly spread and compressed films small domains of the upright lipid population are initially observed, which enlarge with increasing pressure. These domains are no longer existent after 12 h of spreading without compression.

Capsids of Tricorn Protease Studied by Electron Cryomicroscopy

Tricorn protease from the archaeon Thermoplasma acidophilum acts “downstream” of the proteasome; in conjunction with its aminopeptidase cofactors it converts peptides generated by the proteasome into free amino acids. The basic functional unit of Tricorn is a homohexamer of the 121-kDa subunit, 20 of which can assemble further to form an icosahedral capsid with a molecular mass of 14.6 MDa. We have used electron cryomicroscopy to determine the structure of the Tricorn capsids to a resolution of 1.3 nm.

Structure of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum as studied by electron tomography

Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein. We have used electron tomography to obtain low-resolution (2–2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain.

The import pathway of human and Thermoplasma 20S proteasomes into HeLa cell nuclei is different from that of classical NLS-bearing proteins.

Wild-type proteasomes of human erythrocytes and the archaeon Thermoplasma acidophilum compete with each other for transport into nuclei of digitonin-permeabilized HeLa cells in the presence of an energy-regenerating system and rabbit reticulocyte lysate. 'NLS'-mutated Thermoplasma proteasomes were also able to compete with human proteasomes in the same assay, although with lower efficiency. Furthermore, in contrast to the other archaeal and bacterial cell lysates tested, the Thermoplasma cytosol efficiently supported nuclear import of human and Thermoplasma proteasomes. However, the same lysate could barely direct the nuclear transport of BSA-NLSsv40 peptide conjugates or the classical NLS-bearing protein, nucleoplasmin. Finally, additional importin alpha/beta significantly decreased the import efficiency of both human and Thermoplasma proteasomes. Taken together, these results suggest that nuclear import of proteasomes may use a novel pathway that is different from that of classical NLS-bearing proteins.

Calorimetry of archaeal tetraether lipid—indication of a novel metastable thermotropic phase in the main phospholipid from Thermoplasma acidophilum cultured at 59°C

The main glycophospholipid (MPL) from the archaeon Thermoplasma acidophilum is composed of a di-isopranol-2,3-glycerotetraether. The fraction of pentacyclizations of its hydrocarbon chains increases with the growth temperature of the source organism (39 and 59°C), the respective lipids being named MPL39 and MPL59. MPL has a main phase transition between −15 and −30°C. Non-hydrated and hydrated samples of MPL59 have been studied by differential thermal analysis (DTA). Non-hydrated MPL59 does not exert any phase transition. Computer simulation of an unhydrated MPL molecule with four pentacycles and another without pentacyclations demonstrates similar behavior, i.e. the MPL molecules form coils with both polar ends getting closely together. The molecule without pentacyclation coils faster than that with pentacycles. With hydrated samples, DTA scanning conditions were varied. Under certain conditions, the shape of the calorimetric scans, i.e. occurrence of an additional (endotherm) phase transition peak at +17°C and enthalpy changes of the phase transitions indicate a (metastable) solid-analogue phase in MPL59 in addition to the well-known liquid–crystalline phase. Only lipid samples from T. acidophilum with a high degree of acyclic hydrocarbon chains (MPL39) had thus far been reported to form a metastable solid-analogue phase (Blöcher, D., Gutermann, R., Henkel, B., Ring, K., 1990. Biochim. Biophys. Acta 1024, 54–60). A phase transition model is presented for MPL59 which includes the existence of a metastable solid-analogue phase.

Conformational constraints for protein self-cleavage in the proteasome

The proteasome is the central enzyme of protein degradation in the cytosol and the nucleus. It is involved in the removal of abnormal, misfolded or incorrectly assembled proteins, in the processing or degradation of transcriptional regulators in stress response, in degradation of cyclins in cell-cycle control, in the destruction of transcription factors or metabolic enzymes in cell differentiation and metabolic response, and in MHC class I mediated cellular immune response. By the analysis of the crystal and molecular structures of the 20 S proteasomes from the archaeon Thermoplasma acidophilum and from yeast it was shown that the β-type subunits in which the proteolytic activities reside are members of the N-terminal nucleophile (Ntn) protein family. They are synthesized as proproteins and become active by autoprocessing at a Gly - 1-Thr1 bond. The Thr1Ala mutant of subunit β1/Pre3 of the 20 S proteasome from yeast is unable to autolyse. Its crystal and molecular structure at 2.2 Å resolution described here shows that the pro-segment adopts a well-defined γ-turn conformation at Gyl - 1 and provides a first view at an autolysis site in Ntn hydrolases. The Gyl - 1 carbonyl oxygen displays two hydrogen bonds. The modelled Thr1 side-chain is located above the γ-turn bulge such that addition of its nucleophilic hydroxyl group to the electrophilic Gyl - 1 carbonyl carbon atom may proceed by very small motions. The pro-segment binding site and the catalytic site provide a rigid structural framework and appropriate hydrogen bond donors for this reaction. The same structure also supports addition of the Thr1 hydroxyl group to the carbonyl carbon atom of Leu - 2 as a model for the first step in substrate hydrolysis by the proteasome.

Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus.

The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular mass of 42679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95 degrees C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme form the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties of the enzyme.

Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum

A member of the AAA family of Mg 2+-ATPases from the archaeon Thermoplasma acidophilum has been cloned and expressed in Escherichia coli. The protein, VCP-like ATPase of Thermoplasma acidophilum (VAT), is a homologue of SAV from Sulfolobus acidocaldarius and CdcH of Halobacterium salinarium, and belongs to the CDC48/VCP/p97 subfamily. The deduced product of the vat gene is 745 residues long ( M r 83 000), which has an optimal Mg 2+-ATPase activity at 70°C. Electron microscopy shows the purified protein to form single and double homo-hexameric rings. Although the symmetry is different, the appearance of the complexes formed of two rings resembles the 20S proteasome and Hsp60/GroEL. © 1997 Federation of European Biochemical Societies.

The thermosome: alternating α and β-subunits within the chaperonin of the archaeon Thermoplasma acidophilum

The thermosome of the archaeon Thermoplasma acidophilum is composed of two subunits, α and β, which are arranged in two stacked, eight-membered rings. Electron cryo-microscopy in conjunction with image analysis revealed a 4-fold symmetry in the heterooligomeric α+β thermosome isolated from Thermoplasma, but not in the homooligomeric α-only thermosome expressed in Escherichia coli. This indicates that α and β-subunits alternate within the rings of the Thermoplasma thermosome rather than forming two different homooligomeric rings. In addition, a small subpopulation of 9-fold symmetric complexes was found among the recombinant α-only thermosomes, and a central mass most likely representing bound substrate molecules was observed in about half of the native and recombinant thermosome particles.

The Effect of Cysteine-43 Mutation on Thermostability and Kinetic Properties of Citrate Synthase fromThermoplasma acidophilum

In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic ArchaeonThermoplasma acidophilum,for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutantThermoplasmacitrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.

Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: Enzyme purification, characterisation and N-terminal sequence

An NAD(P)-glucose dehydrogenase from the extremely halophilic Archaeon, Haloferax mediterranei, has been purified to electrophoretic homogeneity. The purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability. The N-terminal amino acid sequence has been determined and N-terminus alignment with sequences of other glucose dehydrogenases shows that the halophilic enzyme most closely resembles the NAD(P)-linked glucose dehydrogenase from the thermophilic Archaeon Thermoplasma acidophilum. However, the halophilic glucose dehydrogenase appears to be a dimeric protein, in contrast to the tetrameric enzyme from the thermophile.

Nuclear localization signals of human and Thermoplasma proteasomal alpha subunits are functional in vitro.

Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits. We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (Hsc iota, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct non-nuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both cell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence.

A Proteasome from the Methanogenic Archaeon Methanosarcina thermophila

A 645-kDa proteasome was purified from Methanosarcina thermophila which had chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities and contained alpha (24-kDa) and beta (22-kDa) subunits. Processing of both subunits was suggested by comparison of N-terminal sequences with the sequences deduced from the alpha- and beta-encoding genes (psmA and psmB). Alignment of deduced sequences for the alpha and beta subunits revealed high similarity; however, the N-terminal sequence of the alpha subunit contained an additional 24 amino acids that were not present in the beta subunit. The alpha and beta subunits had high sequence identity with alpha- and beta-type subunits of proteasomes from eucaryotic organisms and the distantly related archaeon Thermoplasma acidophilum. The psmB gene was transcribed in vivo as a monocistronic message from a consensus archaeal promoter. The results suggest that proteasomes are more widespread in the Archaea than previously proposed. Southern blotting experiments suggested the presence of ubiquitin-like sequences in M. thermophila.

EPR Characterization of an Archaeal Succinate Dehydrogenase in the Membrane-Bound State

The membrane-bound succinate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum was characterized by EPR spectroscopy and its functional properties were determined. The highest turnover values of succinate dehydrogenase activity were observed at pH 7.4, which is somewhat above the internal pH value of T. acidophilum. The temperature optimum of the reaction was determined as 78 degrees C and the Km value for succinate using phenazine methosulfate as the electron acceptor at 53 degrees C was 0.32 mM. The membrane-bound enzyme was able to reduce the artificial electron acceptors phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,6-dichloroindophenol. Succinate oxidation was coupled to oxygen consumption in a completely 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive manner. In the oxidized state, T. acidophilum membranes exhibited an almost isotropic EPR spectrum with g-values at gz = 2.017, gy = 2.000, and gx = 1.968 that were assigned to a [3Fe-4S]1+ cluster (S3). Upon reduction with succinate, the membranes displayed a spectrum characteristic of 2Fe-2S clusters (S1), with g-values at gz = 2.029, gy = 1.935, and gx = 1.915. In the dithionite-reduced state, additional resonances can be observed. An axial component, with g-values at gz = 2.057, gy = 1.917, and gx = 1.917 was assigned to a [4Fe-4S]1+ cluster. The saturation behaviour of the S1 cluster was strongly altered in the dithionite-reduced form, thus indicating spin-spin interaction between the S1 center and another paramagnetic center, possibly cluster S2. In both the succinate and the dithionite-reduced membranes, parallel-mode EPR spectra displayed a resonance at g = 14, which may be due to a transition of the S = 2 multiplet of the reduced 3Fe-4S cluster. Spin quantitation yielded a relative stoichiometry of cluster S1 to cluster S3 of 1:1. The results obtained by EPR spectroscopy indicated that the characteristic iron-sulfur cluster S1 [2Fe-2S], S2 [4Fe-4S], and S3 [3Fe-4S], were also present in this archaeal succinate dehydrogenase. EPR redox titrations of T. acidophilum membranes at pH 5.5 yielded a reduction potential of +60 +/- 20 mV for cluster S3 and of +68 +/- 20 mV for cluster S1. The axial [4Fe-4S]2+/1+ center had a reduction potential of -210 +/- 20 mV.

Zinc binding of alcohol and sorbitol dehydrogenases.

Zinc is an essential component of many enzymes, serving a role for catalytic activity or structural stability. The removal of catalytic zinc results in an inactive apoenzyme which, however, often retains the native tertiary structure. Structural zinc frequently contributes to the maintenance of the structure of oligomeric enzymes. The removal of zinc from such proteins therefore prevents subunit association. As discerned from zinc analysis of structurally investigated zinc metalloenzymes, the characteristics of a catalytic zinc-binding motif, in many cases, is a combination of three His/Glu/Asp/Cys residues and an activated H2O-molecule (Vallee & Auld, 1990). The spacers between the first and second ligands are short, typically 1–3 amino acids long (alcohol dehydrogenase and sorbitol dehydrogenase are exceptions with 21–25 residues). The second spacer, longer in nature, separates the second and third ligands by about 20–120 amino acid residues (Vallee & Auld, 1989). The structural zinc is necessary for activity only to the extent that the overall conformation of the enzyme affects its action. It serves as a cross-linking agent to stabilize structures (Berg, 1987). The observed pattern of ligands frequently encompasses four cysteine residues, closely spaced in the linear amino acid sequence (Vallee & Auld, 1990). Sorbitol dehydrogenase (SDH) harbours one catalytic zinc atom per subunit (Jeffery et al., 1984a). Because of the structural relationship between SDH and alcohol dehydrogenase (ADH), two of the three ligands to the zinc could be established early in SDH (Cys44 and His69, Jeffery et al., 1984b). The homology in the area around the third zinc ligand in SDH, though, was low toward ADH. The modelled structure of sheep SDH using the crystal structure of horse ADH as reference, gave the best fit with a glutamic acid residue (Glu 155) as the third zinc ligand (Eklund et al., 1985). In order to establish the exact nature of the third zinc ligand, a set of five different potential ligands were mutated to Ala or Gln, residues not able to ligand zinc, and the proteins were expressed in E. coli with subsequent purification and determination of enzyme activities and zinc contents (Karlsson, 1994). ADH contains two zinc atoms per subunit, one catalytic and one structural (Akeson, 1964; Drum et al., 1969; Eklund et al., 1976). The binding site of the structural zinc atom in ADH involves four cysteine residues at positions 97, 100, 103, and 111. Though it has been designated as structural since long, the exact functions, as well as the question which structural property it maintains, are unclear. To investigate the contribution of each of the second zinc ligands to the overall conformation, we have performed in vitro mutagenesis of class I and III ADH (Jelokova et al., 1994). The ligands were mutated, in separate constructs, to non-zinc liganding counterparts, Ala or Ser. Proteins expressed were found to be labile and therefore, were detectable only from crude extracts upon Western blot analysis. Confirmation of correctly working transcription processes was ascertained by positive Northern blot analyses. The recently published crystal structure of glucose dehydrogenase from the archaeon Thermoplasma acidophilum, reveals structural homology (in spite of low sequence identity) to ADH from horse liver and SDH from sheep liver (John et al., 1994). Glucose dehydrogenase is a tetramer and posesses a structural zinc atom, contained within a loop similar to that of ADH. However, the orientation of this structural loop with respect to the subunit is markedly different from that of ADH. This further illustrates the role of the zinc loop in the quaternary structures of several of the enzymes within the medium- chain dehydrogenase/reductase super-family, MDR (Persson et al. 1994)