Thermoacidophilic Archaeon Thermoplasma Acidophilum Research Articles

Characterisation of the First Archaeal Mannonate Dehydratase from Thermoplasma acidophilum and Its Potential Role in the Catabolism of D-Mannose

Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species.

Identification and characterization of 2-keto-3-deoxy-l-rhamnonate dehydrogenase belonging to the MDR superfamily from the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans: implications to l-rhamnose metabolism in archaea

We identified the non-phosphorylated L-rhamnose metabolic pathway (Rha_NMP) genes that are homologous to those in the thermoacidophilic archaeon Thermoplasma acidophilum in the genome of the thermoacidophilic bacterium Sulfobacillus thermosulfidooxidans. However, unlike previously known 2-keto-3-deoxy-L-rhamnonate (L-KDR) dehydrogenase (KDRDH) which belongs to the short chain dehydrogenase/reductase superfamily, the putative KDRDHs in S. thermosulfidooxidans (Sulth_3557) and T. acidophilum (Ta0749) belong to the medium chain dehydrogenase/reductase (MDR) superfamily. We demonstrated that Sulth_3559 and Sulth_3557 proteins from S. thermosulfidooxidans function as L-rhamnose dehydrogenase and KDRDH, respectively. Sulth_3557 protein is an NAD(+)-specific KDRDH with optimal temperature and pH of 50 °C and 9.5, respectively. The K m and V max values for L-KDR were 2.0 mM and 12.8 U/mg, respectively. Sulth_3557 also showed weak 2,3-butanediol dehydrogenase activity. Phylogenetic analysis suggests that Sulth_3557 and its homologs form a new subfamily in the MDR superfamily. The results shown in this study imply that thermoacidophilic archaea metabolize L-rhamnose to pyruvate and L-lactate by using the MDR-family KDRDH similarly to that of the thermoacidophilic bacterium S. thermosulfidooxidans.

Transfer RNA methyltransferases from Thermoplasma acidophilum, a thermoacidophilic archaeon.

We investigated tRNA methyltransferase activities in crude cell extracts from the thermoacidophilic archaeon Thermoplasma acidophilum. We analyzed the modified nucleosides in native initiator and elongator tRNAMet, predicted the candidate genes for the tRNA methyltransferases on the basis of the tRNAMet and tRNALeu sequences, and characterized Trm5, Trm1 and Trm56 by purifying recombinant proteins. We found that the Ta0997, Ta0931, and Ta0836 genes of T. acidophilum encode Trm1, Trm56 and Trm5, respectively. Initiator tRNAMet from T. acidophilum strain HO-62 contained G+, m1I, and m22G, which were not reported previously in this tRNA, and the m2G26 and m22G26 were formed by Trm1. In the case of elongator tRNAMet, our analysis showed that the previously unidentified G modification at position 26 was a mixture of m2G and m22G, and that they were also generated by Trm1. Furthermore, purified Trm1 and Trm56 could methylate the precursor of elongator tRNAMet, which has an intron at the canonical position. However, the speed of methyl-transfer by Trm56 to the precursor RNA was considerably slower than that to the mature transcript, which suggests that Trm56 acts mainly on the transcript after the intron has been removed. Moreover, cellular arrangements of the tRNA methyltransferases in T. acidophilum are discussed.

Enzymatic and regulatory properties of the trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum

Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, β-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases.

Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger.

Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D-aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D-aldohexoses, especially D-mannose. AldT comprises a unique C-terminal tail motif (residues 247-255) that shuts the active-site pocket of the neighboring subunit. The functional role of the C-terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C-terminal deletion mutants (Delta254, Delta253, Delta252, and Delta249) and two site-specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C-terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Delta249 mutant was also determined. The structure showed that the active-site loops undergo a significant conformational change, which leads to the structural deformation of the substrate-binding pocket.

A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic Euryarchaeon Thermoplasma acidophilum

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40-90 degrees C) and pH (pH 3.5-7) and was maximal at 75 degrees C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl(2), the half-life increased from 15 min to 2 h at 80 degrees C.

Thermal stability and biochemical properties of isocitrate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum

Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T(m), of 82.2 and 84.5 degrees C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened N-terminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70 degrees C. The pH-activity profile was bell-shaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a three-fold reduction in activity when the pH was shifted from the pH-optimum at 7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T(m) of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60 degrees C. The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK(a)s of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme.

Purification and characterization of glycerate kinase from the thermoacidophilic archaeonThermoplasma acidophilum: An enzyme belonging to the second glycerate kinase family

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59°C and pH 2. Along with another thermoacidophilic archaeon,Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher inT. acidophilum than inS. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity fromT. acidophilum cell extracts. TheN-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics withK m values of 0.56 and 0.32 mM forDL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70°C. Of the seven sugars and four phosphate donors tested, onlyDL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn2+, Co2+, Ni2+, Zn2+, Ca2+, and Sr2+, were substituted for Mg2+, the enzyme activities were less than 10% of that obtained in the presence of Mg2+. The amino acid sequence ofT. acidophilum glycerate kinase showed no similarity withE. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

Biosynthesis of Archaeal Membrane Lipids: Digeranylgeranylglycerophospholipid Reductase of the Thermoacidophilic Archaeon Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanyl-sn-glyceryl phosphate (archaetidic acid), which is formed by the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. The reductase activity for the key enzyme in membrane lipid biosynthesis, 2,3-digeranylgeranylglycerophospholipid reductase, was detected in a cell free extract of the thermoacidophilic archaeon Thermoplasma acidophilum. The reduction activity was found in the membrane fraction, and FAD and NADH were required for the activity. The reductase was purified from a cell free extract by ultracentrifugation and four chromatographic steps. The purified enzyme showed a single band at ca. 45 kDa on SDS-PAGE, and catalyzed the formation of archaetidic acid from 2,3-di-O-geranylgeranylglyceryl phosphate. Furthermore, the enzyme also catalyzed the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate analogues such as 2,3-di-O-phytyl-sn-glyceryl phosphate, 3-O-(2,3-di-O-phytyl-sn-glycero-phospho)-sn-glycerol and 2,3-di-O-phytyl-sn-glycero-phosphoethanolamine. The N-terminal 20 amino acid sequence of the purified enzyme was determined and was found to be identical to the sequence encoded by the Ta0516m gene of the T. acidophilum genome. The present study clearly demonstrates that 2,3-digeranylgeranylglycerophospholipid reductase is a membrane associated protein and that the hydrogenation of each double bond of 2,3-digeranylgeranylglycerophospholipids is catalyzed by a single enzyme.

Crystallization and preliminary crystallographic analysis of NAD+-preferring aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum.

The aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a 28 kDa molecular-weight enzyme that catalyzes the oxidation of various aldohexoses, with a preference for NAD+ rather than NADP+ as a cofactor. The recombinant AldT was crystallized using the hanging-drop vapour-diffusion technique at 293 K under several acidic conditions with polyethylene glycol (PEG) and ammonium sulfate as precipitants. Optimization of the initial crystallizations conditions yielded single crystals in solution containing 0.1 M sodium acetate pH 4.6, 18%(w/v) PEG 4000, 0.2 M ammonium sulfate and 15%(v/v) glycerol. An X-ray diffraction data set was collected to a resolution of 2.8 A.

Identification and characterization ofThermoplasma acidophilum 2-keto-3-deoxy-D-gluconate kinase: A new class of sugar kinases

The thermoacidophilic archaeonThermoplasma acidophilum has long been known to utilized-glucosevia the non-phosphorylated Entner-Doudoroff (nED) pathway. We now report the identification of a gene encoding 2-keto-3-deoxy-d-gluconate (KDG) kinase. The discovery of this gene implies the presence of a glycolysis pathway, other than the nED pathway. It was found that Ta0122 in theT. acidophilum genome corresponded to KDG kinase. This enzyme shares no similarity with known KDG kinases, and belongs to a novel class of sugar kinases. Of the five sugars tested only KDG was utilized as a substrate.

The Thermoplasma acidophilum Lon protease has a Ser‐Lys dyad active site

A gene with significant similarity to bacterial Lon proteases was identified during the sequencing of the genome of the thermoacidophilic archaeon Thermoplasma acidophilum. Protein sequence comparison revealed that Thermoplasma Lon protease (TaLon) is more similar to the LonB proteases restricted to Gram-positive bacteria than to the widely distributed bacterial LonA. However, the active site residues of the protease and ATPase domain are highly conserved in all Lon proteases. Using site-directed mutagenesis we show here that TaLon and EcLon, and probably all other Lon proteases, contain a Ser-Lys dyad active site. The TaLon active site mutants were fully assembled and, similar to TaLon wild-type, displayed an apparent molar mass of 430 kDa upon gelfiltration. This would be consistent with a hexameric complex and indeed electron micrographs of TaLon revealed ring-shaped particles, although of unknown symmetry. Comparison of the ATPase activity of Lon wild-type from Thermoplasma or Escherichia coli with respective protease active site mutants revealed differences in Km and V values. This suggests that in the course of protein degradation by wild-type Lon the protease domain might influence the activity of the ATPase domain.

Analysis of Bacterial Glucose Dehydrogenase Homologs from Thermoacidophilic ArchaeonThermoplasma acidophilum: Finding and Characterization of Aldohexose Dehydrogenase

The NADP(+)-preferring glucose dehydrogenase from thermoacidophilic archaeon Thermoplasma acidophilum has been characterized, and its crystal structure has been determined (Structure, 2:385-393, 1994). Its sequence and structure are not homologous to bacterial NAD(P)(+)-dependent glucose dehydrogenases, and its molecular weight is also quite defferent. On the other hand, three functionally unknown genes with homologies to bacterial NAD(P)(+)-dependent glucose dehydrogenases have been sequenced as part of the T. acidophilum genome project (gene names: Ta0191, Ta0747, and Ta0754 respectively). We expressed two genes of three, Ta0191 and Ta0754, in Escherichia coli, and purified the gene products to homogeneity. Dehydrogenase activities were thereby detected from the purified proteins. The Ta0754 gene product exhibited aldohexose dehydrogenase activity, and the Ta0191 gene product exhibited weak 2-deoxyglucose dehydrogenase activity. No aldohexose dehydrogenase gene has been isolated, while the enzyme was reported in 1968. This is the first report of the gene and primary structure. The purified Ta0754 gene product, designated AldT, was characterized. The enzyme AldT effectively catalyzed the oxidation of various aldohexoses, especially D-mannose. Lower activities on D-2-deoxyglucose, D-xylose, D-glucose, and D-fucose were detected although no activities were shown on other aldohexoses or additional sugars. As a cofactor, NAD(+) was much more suitable for the activity than NADP(+). The NAD(+)-preferring dehydrogenase most effectively reacting to D-mannose is for the first time. AldT was most active at pH 10 and above 70 degrees C, and completely stable up to 60 degrees C after incubation for 15 min. Other enzymatic properties were also investigated.

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum.

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [(14)C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [(14)C]mevalonic acid, [(14)C]glycerol, and [(32)P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and beta- l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.

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.

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.

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.