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PubMed1999

Relationship between glycosyl hydrolase inventory and growth physiology of the hyperthermophile Pyrococcus furiosus on carbohydrate-based media.

Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several alpha-linked carbohydrates and cellobiose but not on glucose or other beta-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98 degrees C occurred on beta-1,3- and beta-1,3-beta-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of beta-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98 degrees C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism.

Carbohydrate Metabolism↗
PubMed1999

Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme.

The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed2001

Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi.

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degrees C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degrees C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

Alkaline Phosphatase↗
PubMed2001

Novel bifunctional hyperthermostable carboxypeptidase/aminoacylase from Pyrococcus horikoshii OT3.

Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of Sulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90 degrees C, with the highest activity above 95 degrees C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

Amidohydrolases↗
PubMed2002

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli.

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.

Animals↗
PubMed2003

Heat shock response by the hyperthermophilic archaeon Pyrococcus furiosus.

Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.

Archaeal Proteins↗
PubMed2003

Growth of hyperthermophilic archaeon Pyrococcus furiosus on chitin involves two family 18 chitinases.

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA(BD)), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB(BD) [11 kDa]), followed by a catalytic domain (ChiB(cat) [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.

Amino Acid Sequence↗
PubMed2005

In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus.

A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of -173 mV at 23 degrees C (-193 mV at 80 degrees C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80 degrees C) and low (23 degrees C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

Amino Acid Sequence↗
PubMed2005

First archaeal inorganic polyphosphate/ATP-dependent NAD kinase, from hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization.

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

Adenosine Triphosphate↗
PubMed2006

Flagella of Pyrococcus furiosus: multifunctional organelles, made for swimming, adhesion to various surfaces, and cell-cell contacts.

Pyrococcus furiosus ("rushing fireball") was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100 degrees C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces.

Archaeal Proteins↗
PubMed2006

Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters.

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

ATP-Binding Cassette Transporters↗
PubMed2006

Role of the beta1 subunit in the function and stability of the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus genome encodes three proteasome component proteins: one alpha protein (PF1571) and two beta proteins (beta1-PF1404 and beta2-PF0159), as well as an ATPase (PF0115), referred to as proteasome-activating nucleotidase. Transcriptional analysis of the P. furiosus dynamic heat shock response (shift from 90 to 105 degrees C) showed that the beta1 gene was up-regulated over twofold within 5 minutes, suggesting a specific role during thermal stress. Consistent with transcriptional data, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that incorporation of the beta1 protein relative to beta2 into the 20S proteasome (core particle [CP]) increased with increasing temperature for both native and recombinant versions. For the recombinant enzyme, the beta2/beta1 ratio varied linearly with temperature from 3.8, when assembled at 80 degrees C, to 0.9 at 105 degrees C. The recombinant alpha+beta1+beta2 CP assembled at 105 degrees C was more thermostable than either the alpha+beta1+beta2 version assembled at 90 degrees C or the alpha+beta2 version assembled at either 90 degrees C or 105 degrees C, based on melting temperature and the biocatalytic inactivation rate at 115 degrees C. The recombinant CP assembled at 105 degrees C was also found to have different catalytic rates and specificity for peptide hydrolysis, compared to the 90 degrees C assembly (measured at 95 degrees C). Combination of the alpha and beta1 proteins neither yielded a large proteasome complex nor demonstrated any significant activity. These results indicate that the beta1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role and influences biocatalytic properties, suggesting that beta subunit composition is a factor in archaeal proteasome function during thermal stress, when polypeptide turnover is essential to cell survival.

Archaeal Proteins↗
PubMed1998

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus.

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

Amino Acid Sequence↗
PubMed1999

An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes beta-1,4 bonds in mixed-linkage (1-->3),(1-->4)-beta-D-glucans and cellulose.

The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed beta-1,4 but not beta-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only beta-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage beta-glucans such as barley beta-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (-I, -II, and -III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100 degreesC and 6.0, respectively; a half-life of 40 h at 95 degreesC; and a denaturing temperature of 112 degreesC as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.

Amino Acid Sequence↗
PubMed1999

Transcriptional regulation in the hyperthermophilic archaeon Pyrococcus furiosus: coordinated expression of divergently oriented genes in response to beta-linked glucose polymers.

The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular beta-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular beta-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the beta-1,4-linked glucose dimer cellobiose or the beta-1,3-linked glucose polymer laminarin, the activities of both beta-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.

Alcohol Dehydrogenase↗
PubMed1999

A hyperactive NAD(P)H:Rubredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus.

NAD(P)H:rubredoxin oxidoreductase (NROR) has been purified from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is exceedingly active in catalyzing the NADPH-dependent reduction of rubredoxin, a small (5.3-kDa) iron-containing redox protein that had previously been purified from this organism. The apparent Vmax at 80 degrees C is 20,000 micromol/min/mg, which corresponds to a kcat/Km value of 300,000 mM(-1) s(-1). The apparent Km values measured at 80 degrees C and pH 8.0 for rubredoxin, NADPH, and NADH were 50, 5, and 34 microM, respectively. The enzyme did not reduce P. furiosus ferredoxin. NROR is a monomer with a molecular mass of 45 kDa and contains one flavin adenine dinucleotide molecule per mole but lacks metals and inorganic sulfide. The possible physiological role of this hyperactive enzyme is discussed.

Amino Acid Sequence↗
PubMed1999

Acetyl coenzyme A synthetase (ADP forming) from the hyperthermophilic Archaeon pyrococcus furiosus: identification, cloning, separate expression of the encoding genes, acdAI and acdBI, in Escherichia coli, and in vitro reconstitution of the active heterotetrameric enzyme from its recombinant subunits.

Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) --> acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile Pyrococcus furiosus. Isoform I is a heterotetramer (alpha(2)beta(2)) with an apparent molecular mass of 145 kDa, composed of two subunits, alpha and beta, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated acdAI and acdBI, were identified in the genome of P. furiosus. The genes were separately overexpressed in Escherichia coli, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from P. furiosus.

Amino Acid Sequence↗
PubMed1999

An unusual oxygen-sensitive, iron- and zinc-containing alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degreesC by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 +/- 1,000, appears to be a homohexamer, and contains iron ( approximately 1.0 g-atom/subunit) and zinc ( approximately 1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23 degreesC. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95 degreesC of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9. 4 and 10.2 (at 80 degreesC), and the apparent Kms (at 80 degreesC) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.

Alcohol Dehydrogenase↗