Characterization of an Ancestral Type of Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophilic Bacterium, Thermotoga maritima

Blamey, Jenny M.; Adams, Michael W. W.

doi:10.1021/bi00170a019

Cited by 108 publications

(82 citation statements)

References 32 publications

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“…The Arrhenius plot showed a pronounced transition point at 60°C, which has been observed for several other enzymes of Pyrococcus (Blarney and Adams, 1994;Kengen et al, 1995). The enzyme showed high thermostability in the presence of salts, which has been shown for other enzymes from several hyperthermophiles, e.g.…”

Section: Discussionsupporting

confidence: 69%

Purification and Properties of Acetyl‐CoA Synthetase (ADP‐forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus

Glasemacher

Bock

Schmid

et al. 1997

European Journal of Biochemistry

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Acetyl-CoA synthetase (ADP-forming) is an enzyme in Archaea that catalyzes the formation of acetate from acetyl-CoA and couples this reaction with the synthesis of ATP from ADP and P, (acetyl-CoA + ADP + P, -acetate + ATP + CoA) [Schafer, T., Selig, M. & Schonheit, P. (1993) Arch. Microbiol. 159, 72-83]. The enzyme from the anaerobic hyperthermophile Pyrococcus furiosus was purified 96-fold with a yield of 20% to apparent electrophoretic homogeneity. The oxygen-stable enzyme had an apparent molecular mass of 145 kDa and was composed of two subunits with apparent molecular masses of 47 kDa and 25 kDa, indicating an a2,B2 structure. The N-terminal amino acid sequences of both subunits were determined; they do not show significant identity to other proteins in databases. The purified enzyme catalyzed the reversible conversion of acetyl-CoA, ADP and P, to acetate, ATP and CoA. The apparent V,,, value in the direction of acetate formation was 18 U/mg (55 "C), the apparent K,,, values for acetylCoA, ADP and P, were 17 pM, 60 pM and 200 FM, respectively. ADP and P, could not be replaced by AMP and PP,, defining the enzyme as an ADP-forming rather than an AMP-forming acetyl-CoA synthetase. The apparent V,,, value in the direction of acetyl-CoA formation was about 40 U/mg (55"C), and the apparent K, values for acetate, ATP and CoA were 660 pM, 80 pM and 30 pM, respectively. The purified enzyme was not specific for acetyl-CoA or acetate, in addition to acetyl-CoA (loo%), the enzyme accepts propionyl-CoA (110%) and butyryl-CoA (92%), and in addition to acetate (100%), the enzyme accepts propionate (loo%), butyrate (92%), isobutyrate (79 %), valerate (36%) and isovalerate (34%), indicating that the enzyme functions as an acyl-CoA synthetase (ADP-forming) with a broad substrate spectrum. Succinate, phenylacetate and indoleacetate did not serve as substrates for the enzyme (< 3 %). In addition to ADP (loo%), GDP (220%) and IDP (250%) were used, and in addition to ATP (loo%), GTP (210%) and ITP (320%) were used. Pyrimidine nucleotides were not accepted. The enzyme was dependent on Mg", which could be partly substituted by Mn2+ and Co2+. The pH optimum was pH 7. The enzyme has a temperature optimum at 90"C, which is in accordance with its physiological function under hyperthermophilic conditions. The enzyme was stabilized against heat inactivation by salts. In the presence of KCI (1 M), which was most effective, the enzyme did not loose activity after 2 h incubation at 100°C.Keywords: acetyl-CoA synthetase (ADP-forming) ; acetate formation; Archaea; Pyrococcus furiosus; hyperthermophiles.Acetyl-CoA synthetase (ADP-forming) catalyzes the following reversible reaction: acetyl-CoA + ADP + Pi * acetate + ATP + CoA. This unusual synthetase was first detected in the eukaryotic parasite Entamoeba histolytica and later in the anaerobic protozoon Giardia lamblia, where it is involved in acetate formation and ATP production during fermentative metabolism (Reeves et al., 1977;Lindmark, 1980; reviewed by Muller, 1988;Adam, 1991).In prok...

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Section: Discussionsupporting

confidence: 69%

Purification and Properties of Acetyl‐CoA Synthetase (ADP‐forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus

Glasemacher

Bock

Schmid

et al. 1997

European Journal of Biochemistry

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“…According to the present EPR data, this exchange should not be limited to thiolate exchange but could involve other functional groups [23,24]. This situation is somewhat reminiscent of that observed with Thermotoga maritima PFO [17] for which three EPR signals were detected, although other data pointed to only two [4Fe~S] clusters per mole. However, one of the EPR signals involved in Thermotoga maritima PFO appeared upon CoASH addition alone [18], whereas no signal was detected in Clostridium pasteurianum PFO under such conditions.…”

Section: Discussionsupporting

confidence: 76%

“…In C. pasteurianum PFO, as in other such enzymes thought to contain two [4Fe~S] clusters per subunit [17,20], pyruvate does not generate the EPR signal of a radical. In contrast, an EPR signal assigned to the hydroxyethyl-TPP radical [4,18,22] appears upon addition of pyruvate to PFO containing an odd (one or three) number of [4Fe~S] clusters.…”

Section: Discussionmentioning

confidence: 95%

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Molecular mechanism of pyruvate‐ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme

et al. 1996

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“…All assays were carried out under anaerobic conditions at 80ЊC. The activities of POR, IOR, KGOR, and 2-ketoisovalerate oxidoreductase (VOR) were determined by measuring the substrate-dependent reduction of methyl viologen in the presence of CoA with pyruvate, indolepyruvate, 2-ketoglutarate, or 2-ketoisovalerate as the substrate (final concentration, 5 mM), respectively (11,45). AOR, FOR, and FMOR activities were measured by the CoA-independent reduction of benzyl viologen.…”

Section: Methodsmentioning

confidence: 99%

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

et al. 1995

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The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91؇C by the fermentation of peptides and reduces elemental sulfur (S 0 ) to H 2 S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S 0 in the medium, and no growth was observed in the absence of S 0 . The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S 0 concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S 0 limitation. This was accompanied by increased amounts of H 2 and alcohol (ethanol and butanol) from cultures grown with limiting S 0 . Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M r of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under S 0 limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.Hyperthermophiles are a recently discovered group of microorganisms that have the remarkable property of growing at temperatures of 90ЊC and above (1, 2, 64, 65). They have been isolated from a variety of geothermally heated environments, and almost all are classified as Archaea (formerly Archaeobacteria [75]). The majority are strict anaerobes, and most are obligately dependent upon the reduction of elemental sulfur (S 0 ) to H 2 S for optimal growth. Various organic compounds and molecular H 2 serve as electron donors for the apparent respiration of S 0 . The mechanism of S 0 reduction in one autotrophic hyperthermophile, Pyrodictium brockii, an obligate S 0 -reducing species which grows optimally at 105ЊC, has been investigated. This organism was shown to have a primitive membrane-bound electron transport chain for coupling H 2 oxidation and H 2 S production (52), although the S 0 -reducing entity was not purified.On the other hand, some of the heterotrophic hyperthermophiles are able to grow well in the absence of S 0 . These have fermentative-type metabolisms and produce H 2 during growth, although they also produce H 2 S if S 0 is added to the growth medium. S 0 reduction was thought to be...

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Characterization of an Ancestral Type of Pyruvate Ferredoxin Oxidoreductase from the Hyperthermophilic Bacterium, Thermotoga maritima

Cited by 108 publications

References 32 publications

Purification and Properties of Acetyl‐CoA Synthetase (ADP‐forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus

Purification and Properties of Acetyl‐CoA Synthetase (ADP‐forming), an Archaeal Enzyme of Acetate Formation and ATP Synthesis, from the Hyperthermophile Pyrococcus furiosus

Molecular mechanism of pyruvate‐ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

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