Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S0 is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S0 reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (M r, 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S0 addition. This primary response to S0 also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S0 addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S0. A secondary response to S0 is observed 30 min after S0 addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S0-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S0-reducing system in autotrophic archaea and bacteria.

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Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic ArchaeonPyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.183.2.716-724.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 716-724 ◽

Cited By ~ 114

Author(s):

Michael W. W. Adams ◽

James F. Holden ◽

Angeli Lal Menon ◽

Gerrit J. Schut ◽

Amy M. Grunden ◽

...

Keyword(s):

Growth Rates ◽

Elemental Sulfur ◽

Pyrococcus Furiosus ◽

Growth Conditions ◽

Growth Media ◽

Hyperthermophilic Archaeon ◽

Order Of Magnitude ◽

Membrane Bound ◽

Peptide Metabolism ◽

Specific Activities

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosusgrows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S0). Growth rates were highest on media containing peptides and S0, with or without maltose. Growth did not occur on the peptide medium without S0. S0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S0 with or without maltose were the same, suggesting that S0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S0-reducing enzyme in this organism and the mechanism of the S0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

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Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.00123-10 ◽

2010 ◽

Vol 76 (18) ◽

pp. 6286-6289 ◽

Cited By ~ 36

Author(s):

Jae Kyu Lim ◽

Sung Gyun Kang ◽

Alexander V. Lebedinsky ◽

Jung-Hyun Lee ◽

Hyun Sook Lee

Keyword(s):

Gene Cluster ◽

In Silico ◽

In Silico Analysis ◽

Sequence Motifs ◽

Thermococcus Onnurineus Na1 ◽

Hyperthermophilic Archaeon ◽

Group 4 ◽

Membrane Bound ◽

Silico Analysis

ABSTRACT In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs.

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The Elemental Sulfur-Responsive Protein (SipA) from the Hyperthermophilic Archaeon Pyrococcus furiosus Is Regulated by Sulfide in an Iron-Dependent Manner

Journal of Bacteriology ◽

10.1128/jb.00660-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5841-5843 ◽

Cited By ~ 6

Author(s):

Sonya M. Clarkson ◽

Elizabeth C. Newcomer ◽

Everett G. Young ◽

Michael W. W. Adams

Keyword(s):

Elemental Sulfur ◽

Iron Sulfide ◽

Pyrococcus Furiosus ◽

Protein A ◽

Dependent Manner ◽

Intracellular Iron ◽

Hyperthermophilic Archaeon

ABSTRACT The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S0). Expression of sipA is regulated by sulfide, the product of S0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

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Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

Journal of Bacteriology ◽

10.1128/jb.181.11.3358-3367.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3358-3367 ◽

Cited By ~ 54

Author(s):

Karina B. Xavier ◽

Ralf Peist ◽

Marina Kossmann ◽

Winfried Boos ◽

Helena Santos

Keyword(s):

Specific Activity ◽

Pyrococcus Furiosus ◽

Maximal Activity ◽

Consensus Binding Site ◽

Calculated Molecular Mass ◽

Thermococcus Litoralis ◽

Hyperthermophilic Archaeon ◽

Key Enzymes ◽

Maltose Metabolism ◽

Cell Extracts

ABSTRACT Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected inT. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, ap-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. AKm of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacteriumThermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

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Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.187.6.2077-2083.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 2077-2083 ◽

Cited By ~ 18

Author(s):

Sherry V. Story ◽

Claudia Shah ◽

Francis E. Jenney ◽

Michael W. W. Adams

Keyword(s):

Metal Ion ◽

Specific Activity ◽

Pyrococcus Furiosus ◽

High Specific Activity ◽

Native Form ◽

Genome Database ◽

Hyperthermophilic Archaeon ◽

Cell Extracts ◽

Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

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Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase

Journal of Bacteriology ◽

10.1128/jb.186.23.7888-7895.2004 ◽

2004 ◽

Vol 186 (23) ◽

pp. 7888-7895 ◽

Cited By ~ 68

Author(s):

Michael V. Weinberg ◽

Francis E. Jenney ◽

Xiaoyuan Cui ◽

Michael W. W. Adams

Keyword(s):

Hydrogen Peroxide ◽

Electron Donor ◽

Metal Content ◽

Specific Activity ◽

Pyrococcus Furiosus ◽

Hyperthermophilic Archaeon ◽

E Coli ◽

Reducing Conditions ◽

Metal Contents ◽

Protein Functions

ABSTRACT Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 ± 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85°C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 μmol of rubredoxin oxidized/min/mg with apparent Km values of 35 and 70 μM for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 μmol of H2O2 reduced/min/mg of rubrerythrin at 85°C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide:rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

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Cellobiose Uptake in the Hyperthermophilic ArchaeonPyrococcus furiosus Is Mediated by an Inducible, High-Affinity ABC Transporter

Journal of Bacteriology ◽

10.1128/jb.183.17.4979-4984.2001 ◽

2001 ◽

Vol 183 (17) ◽

pp. 4979-4984 ◽

Cited By ~ 31

Author(s):

Sonja M. Koning ◽

Marieke G. L. Elferink ◽

Wil N. Konings ◽

Arnold J. M. Driessen

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Abc Transporter ◽

Transport System ◽

Abc Transporters ◽

Pyrococcus Furiosus ◽

Binding Protein ◽

Hyperthermophilic Archaeon ◽

High Affinity ◽

Dependent Transport

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosuscan utilize different β-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K m = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosusmembranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtAgene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

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An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.181.4.1163-1170.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1163-1170 ◽

Cited By ~ 37

Author(s):

Kesen Ma ◽

Michael W. W. Adams

Keyword(s):

Alcohol Dehydrogenase ◽

Specific Activity ◽

Pyrococcus Furiosus ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Physiological Role ◽

Resonance Spectroscopy ◽

Hyperthermophilic Archaeon ◽

Subunit Molecular Weight ◽

Cell Extracts ◽

Oxygen Sensitive

ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C 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 (∼1.0 g-atom/subunit) and zinc (∼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°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent Km s (at 80°C) 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.

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