Pyruvate : ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments

Kunow, Jasper; Linder, Dietmar; Thauer, Rudolf K.

doi:10.1007/s002030050166

Cited by 22 publications

(33 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The enzyme showed high thermostability in the presence of salts, which has been shown for other enzymes from several hyperthermophiles, e.g. Methanopyrus kandleri and Archaeoglobus fulgidus (Breitung et al, 1991(Breitung et al, , 1992Ma et al, 1991;Kunow et al, 1995;reviewed by Adams, 1993 ;Leuschner and Antranikian, 1995). Acetyl-CoA synthetase (ADP-forming) of f?…”

Section: Discussionsupporting

confidence: 52%

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

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 52%

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

View full text Add to dashboard Cite

show abstract

“…Another possibility would be that these POR might be differentially expressed at different growth temperatures. While the 4 subunit type of POR seems to be most common in hyperthermophiles [33], its presence has also been observed in mesophiles [34]. Conversely the single subunit type, also found in the mesophile C. acetobutylicum has an in vitro temperature optimum of 60°C [21].…”

Section: Discussionmentioning

confidence: 98%

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

et al. 2008

View full text Add to dashboard Cite

Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H 2 and CO 2 . Very little is known about the factors that determine metabolic fl uxes infl uencing H 2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identifi ed genes encoding key enzymes in pyruvate catabolism and H 2 -synthesis pathways, and have confi rmed transcription of these genes throughout growth on α-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as "Energy-converting Hydrogenases", or via NAD(P)Hdependent Fe-only hydrogenases which would permit H 2 production from NADH generated during the glyceraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our fi ndings show the presence of a gene cluster putatively encoding a membrane integral NADH:Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H 2 synthesis is the fi rst step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H 2 synthesis remain to be elucidated, and will need to be confi rmed using both expression analysis and protein characterization.

show abstract

“…2 suggest that this is also true for T. litoralis. From recent molecular analyses (32), it was concluded that the PORs in mesophilic organisms (11,30,43,57,62,63,67), most of which contain a single large subunit (14,27,32,49), originated from four ancestral genes of the type now present in the hyperthermophilic 2-ketoacid oxidoreductases (8,32,34). VOR is especially interesting in this respect because of its broad substrate specificity, suggesting that it is perhaps even more closely related than POR or KGOR to the putative ancestral oxidoreductase.…”

Section: Por and Kgor Comprise Four Different Subunits (␣␤␥␦) Withmentioning

confidence: 99%

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea

1996

View full text Add to dashboard Cite

Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M r of ca. 230,000, with subunit M r values of 47,000 (␣), 34,000 (␤), 23,000 (␥), and 13,000 (␦). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (␣␤␥␦), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (k cat /K m > 1.0 M ؊1 s ؊1; K m < 100 M) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (k cat /K m < 0.2 M ؊1 s ؊1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85؇C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent K m , 250 M) and CO 2 (apparent K m , 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.In the last decade, a remarkable group of microorganisms capable of growing at temperatures around 100ЊC have been isolated from geothermally heated habitats (59,60). Virtually all of these so-called hyperthermophiles are classified as archaea (formerly archaebacteria [68]). Most of them are obligately anaerobic organisms and obtain energy for growth by either fermentation, sulfate reduction, or methanogenesis (1, 2, 28). The fermentative hyperthermophiles typically require elemental sulfur (S 0 ), which is reduced to H 2 S, for growth, although some, such as species of Pyrococcus and Thermococcus, grow well in the absence of S 0 (22,38,48). A characteristic of these organisms is their ability to ferment peptides, and some can also utilize carbohydrates (28,59,60). The pathways of carbohydrate fermentation have recently been shown to resemble the well-known bacterial pathways but with some unique additions and possibly a combination of different pathways (29,46,56).Only limited information is available on the degradation of amino acids by hyperthermophilic archaea (2, 28). Several of these organisms have been shown to contain high protease (9, 21, 33), glutamate dehydrogenase (16,19,40,54) and aromatic amino acid transaminase (3, 4) activities, and these enzymes have been purified from one or more species. In addition, three ...

show abstract

Pyruvate : ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments

Cited by 22 publications

References 0 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

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea

Contact Info

Product

Resources

About