Purification and spectral characterization of a b-type cytochrome from the plasma membrane of the archaebacterium Sulfolobus acidocaldarius

For the first time the purification of a heme-b containing cytochrome from the plasma membrane of an extremely thermoacidophilic archaebacterium is described. The detergent solubilized 30 kDa polypeptide contains two heme-b centers and one copper ion. According to its low temperature spectra and CO-binding properties, it is likely to function as a cytochrome-o like terminal oxidase in the membrane. The purified cytochrome does not retain catalytic activity, however.     

Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius

To investigate how hyperthermophilic archaea can propagate their genomes accurately, we isolated Sulfolobus acidocaldarius mutants exhibiting abnormally high rates of spontaneous mutation. Our isolation strategy involved enrichment for mutator lineages via alternating selections, followed by screening for the production of spontaneous, 5-fluoro-orotate-resistant mutants in micro-colonies. Several candidates were evaluated and found to have high frequencies of pyrE and pyrF mutation and reversion. Neither an increased efficiency of plating of mutants on selective medium, nor the creation of a genetically unstable pyrE allele, could be implicated as the cause of these high frequencies. The strains had elevated frequencies of other mutations, and exhibited certain phenotypic differences among themselves. A large increase in sensitivity to DNA-damaging agents was not observed, however. These properties generally resemble those of bacterial mutator mutants and suggest loss of functions specific to genetic accuracy.     

Characterization and purification of a membrane-bound archaebacterial pyrophosphatase from Sulfolobus acidocaldarius.

Plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) display a pyrophosphate-hydrolyzing activity [M. Lübben & G. Sch?fer (1987) Eur. J. Biochem. 164, 533-540]. In our present work, we solubilized and purified this pyrophosphatase to homogeneity. It consists of a single subunit with a molecular mass of 17-18 kDa, forming an oligomer of 70 kDa under native conditions. Edman degradation revealed 30 amino acids of the N-terminus. The enzyme cleaves phosphoric-acid-anhydride bonds independently of monovalent or divalent cations. Temperature and pH optima of 75 degrees C and 3.5-3.7, respectively, characterize it as an ectoenzyme. Membrane lipids of Sulfolobus stimulate the activity. The dolichol-pyrophosphate-complexing peptide-antibiotic bacitracin inhibited growth of Sulfolobus. A possible function of the acid pyrophosphatase is the hydrolysis of dolichol pyrophosphate in connection with glycosylation reactions of membrane proteins.     

Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.     

Purification of glycerol dialkyl nonitol tetraether from Sulfolobus acidocaldarius

A modified procedure for extraction and purification of hydrolyzed archaebacterial lipids is described. Lipids were extracted from Sulfolobus acidocaldarius using a Soxhlet extraction procedure followed by trichloroacetic acid solvent-extraction of the residue. The yield of total extractable material by this protocol was 14% which, after a two-phase wash, yielded 10% lipid. Modifications to the published steps for purifying the subsequently hydrolyzed lipids were developed to purify glycerol dialkyl nonitol tetraether (GDNT). The nearly colorless final macrocyclic product was characterized by TLC, IR, NMR, and mass spectrometry.     

Cytochrome aa3 from Sulfolobus acidocaldarius. A single-subunit, quinol-oxidizing archaebacterial terminal oxidase

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 369) extrudes protons when expending respiratory energy [Moll, R. & Sch?fer, G. (1988) FEBS Lett. 232, 359-363]. Cytochromes of the membrane electron-transport systems are assumed to represent the proton pumps. Only a- and b-type cytochromes can be found; no c-type cytochromes are present. Of the two terminal oxidases [Anemüller, S. & Sch?fer, G. (1989) FEBS Lett. 244, 451-455] one shows an absorption band at 604-605 nm, typical of cytochromes of the aa3 type. This hemoprotein has been solubilized from the membrane and purified to homogeneity. It exhibits distinct differences from known aa3-type oxidases. (a) It consists of a single polypeptide subunit of 38-40 kDa apparent molecular mass with two heme-a molecules and two copper ions. (b) In the oxidized state, absorption maxima are found at 421 nm and 597 nm, and in the reduced state at 439 nm and 601 nm; CO difference spectra suggest one heme to be a heme-a3 centre. (c) The redox potentials of the heme centres are +220 mV and +370 mV, respectively. (d) A high-spin heme signal at g = 6 is present in EPR spectra, which is more prominent than the low-spin heme signal at g = 3, the former already being present in the oxidized state. A signal at g = 2.1 may be due to one of the copper ions and is superimposed upon a minor free radical signal at g = 2. (e) Caldariella quinone was also isolated from the plasma membrane of Sulfolobus. Its redox midpoint potential at pH 6.5 was determined to be +100 (+/- 5) mV; spectral properties have also been determined. (f) The isolated aa3 preparation does not oxidize cytochrome c; however, it oxidizes N,N,N',N'-tetramethyl-1,4-phenylenediamine dihydrochloride as an artificial single-electron donor as well as reduced caldariella quinone, which is assumed to represent the natural substrate. The reaction is cyanide-sensitive and the product of oxygen reduction is water. (g) On the basis of the results obtained a novel type of cytochrome aa3 is postulated in this paper which oxidizes reduced quinones; its ability to act as a proton pump remains to be shown.     

Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius.

DNA polymerase has been purified about 25,000-fold from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. On SDS-PAGE the enzyme was observed to have a molecular weight of 100 kDa and to be about 90% pure. The native molecular weight was 108 kDa indicating that the enzyme is composed of a single polypeptide. Activity gel analysis showed an active polypeptide of about 100 kDa. Under conditions promoting proteolysis this polypeptide was degraded to a slightly smaller form of 98 kDa. The enzyme has been characterized in respect to optimal assay conditions, template specificity, sensitivity to inhibitors and associated nuclease activities. The high temperature optimum of 65 degrees C should be emphasized. No substantial similarities have been found with other prokaryotic and eukaryotic DNA polymerases, although the enzyme bears certain resemblances to prokaryotic non-replicative polymerases.     

Partial Purification and Characterization of Thermostable Acid Phosphatase from Thermoacidophilic Archaeon Sulfolobus acidocaldarius

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70°C, respectively. The apparent K _m value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75°C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein. Received: 8 July 1999 / Accepted: 9 August 1999     

Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.     

Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a Mr of about 270,000 and was shown to be a hexamer of subunit Mr of 44,000. It was active from 30 to 95 degrees C, with a maximum activity at 85 degrees C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90 degrees C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.     

Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.     

Crystalline NAD/NADP-dependent malate dehydrogenase; the enzyme from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

Malate dehydrogenase from Sulfolobus acidocaldarius has been purified 240-fold to apparent electrophoretic homogeneity. The enzyme shows a specific activity of 277 U/mg and crystallizes readily. The relative molecular mass of the native enzyme is estimated as 128,500 by ultracentrifugation. After cross-linking a relative molecular mass of 134,000 is found by sodium dodecyl sulfate gel electrophoresis. Malate dehydrogenase from S. acidocaldarius is composed of four subunits of identical size with a relative molecular mass of 34,000. Active-enzyme sedimentation in the analytical ultracentrifuge indicates that the tetramer is the catalytically active species. Kinetic studies in the direction of oxaloacetate reduction showed a Km for NADH of 4.1 microM and a Km for oxaloacetate of 52 microM. Oxaloacetate exhibits substrate inhibition at higher concentrations, L-malate, NAD and NADP were found to be product inhibitors. The enzymatic activity is inhibited by 2-oxoglutarate but not by the adenosine nucleotides AMP, ADP and ATP. Only low activity is detected in the direction of malate oxidation. Malate dehydrogenase from S. acidocaldarius utilizes both NADH and NADPH to reduce oxaloacetate. The enzyme shows A-side stereospecificity for both nicotinamide dinucleotides.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase.

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.     

A sulfonolipid and novel glucosamidyl glycolipids from the extreme thermoacidophile Bacillus acidocaldarius.

The total lipid content of the extreme thermoacidophile Bacillus acidocaldarius comprises about 8.1% of the cell dry weight. Total lipid had a distribution of 15.7% neutral linique component initially characterized as an N-acylglucosamine beta-linked to the primary hydroxyl of an unusual fully saturated pentacyclic triterpene derived tetrol(C35H62O4, Mr 546), which appears to be a derivative of the pentacylcic triterpene hopane substituted at C-29 with a 1,2,3,4-tetrahydroxy pentane. Other major glycolipids present were partially characterized as O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta D-glucopyranosyldiacylglycerol and O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylmonoacylglycerol. Minor components of the glycolipid fraction included O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylglycerol, O-2-amino-2-deoxy-beta-D-glucopyranosyl pentacyclic tetrol and free pentacyclic tetrol. The distributions of esterified and amide-linked fatty acids were similar, being comprised primarily of branched heptadecanoic, 11-cyclohexyundecanoic and 13-cyclohexyltridecanoic acids. The acid lipids were composed of a sulfonoglycosyldiacylglycerol (43.2%), diphosphatidylglycerol (32.3%), lysodiphosphatidylglycerol (5.3%), phosphatidic acid (5.8%) and phosphatidylglycerol (13.4%).     

A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus

The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.     

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase.

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Sch?fer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.     

Purification and properties of NAD(P)-independent polyol dehydrogenase complex from the plasma membrane of Gluconobacter oxydans

Gluconobacter oxydans rapidly oxidizes many different polyhydroxy alcohols (polyols). Polyol oxidations are catalyzed by constitutively synthesized membrane-bound dehydrogenases directly linked to the electron transport chain. A polyol-oxidizing enzyme was isolated from the membranes of G. oxydans and tested for its ability to oxidize various substrates. The enzyme was composed of three subunits: a 67 kDa catalytic unit, a 46 kDa c-type cytochrome, and a 15 kDa subunit. The enzyme oxidized compounds containing three or more hydroxyl groups but did not oxidize mono-, di-, or cyclic alcohols; aldehydes; carboxylic acids; or mono- or di-saccharides. Therefore, we propose this enzyme be considered a polyol dehydrogenase.