[11] Aldehyde Oxidoreductases from Pyrococcus furiosus

2001 ◽  
pp. 132-144 ◽  
Author(s):  
Roopali Roy ◽  
Angeli L. Menon ◽  
Michael W.W. Adams
Keyword(s):  
Pyrococcus Furiosus

Electrochemical characterization of a truncated hydrogenase from Pyrococcus furiosus

2021 ◽  
pp. 138502
Author(s):  
Yuanming Wang ◽  
Yunhong Song ◽  
Chunling Ma ◽  
Hong-qi Xia ◽  
Ranran Wu ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Electrochemical Characterization

scholarly journals Glyceraldehyde-3-Phosphate Ferredoxin Oxidoreductase from Methanococcus maripaludis

2007 ◽  
Vol 189 (20) ◽  
pp. 7281-7289 ◽  
Author(s):  
Myong-Ok Park ◽  
Taeko Mizutani ◽  
Patrik R. Jones
Keyword(s):  
Pyrococcus Furiosus ◽  
Substrate Inhibition ◽  
Physiological Role ◽  
Sodium Molybdate ◽  
Rare Metal ◽  
Growth Conditions ◽  
Defined Medium ◽  
Methanococcus Maripaludis ◽  
Ferredoxin Oxidoreductase ◽  
Gapdh Activity

ABSTRACT The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPORPf), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPORMm) was purified from Escherichia coli grown in minimal medium containing 100 μM sodium molybdate. In contrast, GAPORMm obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPORMm was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPORMm catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPORMm is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 μM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPORMm in M. maripaludis most likely involves only nonoptimal growth conditions.

A distinct structure of Cas1–Cas2 complex provides insights into the mechanism for the longer spacer acquisition in Pyrococcus furiosus

2021 ◽  
Vol 183 ◽  
pp. 379-386
Author(s):  
Dongmei Tang ◽  
Huijuan Li ◽  
Chengyong Wu ◽  
Tingting Jia ◽  
Haihuai He ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Distinct Structure

scholarly journals Cell-Free Transcription at 95°: Thermostability of Transcriptional Components and DNA Topology Requirements of Pyrococcus Transcription

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1325-1333
Author(s):  
Carina Hethke ◽  
Agnes Bergerat ◽  
Winfried Hausner ◽  
Patrick Forterre ◽  
Michael Thomm
Keyword(s):  
Transcription Factors ◽  
Half Life ◽  
Pyrococcus Furiosus ◽  
Polymerase Activity ◽  
Supercoiled Dna ◽  
Eukaryotic Transcription ◽  
Transcription System ◽  
Archaeal Transcription ◽  
Protein Components

Abstract Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100°. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100°. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg2+ ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95° was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70° and 90°. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.

scholarly journals In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

2005 ◽  
Vol 71 (3) ◽  
pp. 1522-1530 ◽  
Author(s):  
Amy M. Grunden ◽  
Francis E. Jenney ◽  
Kesen Ma ◽  
Mikyoung Ji ◽  
Michael V. Weinberg ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Pyrococcus Furiosus ◽  
Expression System ◽  
Anaerobic Bacteria ◽  
Flavin Mononucleotide ◽  
Superoxide Reductase ◽  
Recombinant Enzymes ◽  
Significant Similarity ◽  
Midpoint Potential

ABSTRACT 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°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°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.

Comparison of Small- and Large-scale Expression of Selected Pyrococcus furiosus Genes as an Aid to High-throughput Protein Production

2005 ◽  
Vol 6 (2-3) ◽  
pp. 149-158 ◽  
Author(s):  
Frank J. Sugar ◽  
Francis E. Jenney ◽  
Farris L. Poole ◽  
Phillip S. Brereton ◽  
Michi Izumi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Protein Production ◽  
Pyrococcus Furiosus

Three-Dimensional Electron Microscopy of the Clamp Loader Small Subunit from Pyrococcus furiosus

2001 ◽  
Vol 134 (1) ◽  
pp. 35-45 ◽  
Author(s):  
Kouta Mayanagi ◽  
Tomoko Miyata ◽  
Takuji Oyama ◽  
Yoshizumi Ishino ◽  
Kosuke Morikawa
Keyword(s):  
Electron Microscopy ◽  
Pyrococcus Furiosus ◽  
Three Dimensional ◽  
Small Subunit ◽  
Clamp Loader ◽  
Dimensional Electron

scholarly journals Calcium-induced tertiary structure modifications of endo-β-1,3-glucanase from Pyrococcus furiosus in 7.9 M guanidinium chloride

2005 ◽  
Vol 386 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Roberta CHIARALUCE ◽  
Giulio GIANESE ◽  
Sebastiana ANGELACCIO ◽  
Rita FLORIO ◽  
Johan F. T. van LIESHOUT ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Pyrococcus Furiosus ◽  
Glycoside Hydrolases ◽  
Guanidinium Chloride ◽  
Absorption Region ◽  
Native Form ◽  
Carboxylate Groups ◽  
The Family ◽  
Notable Increase ◽  
Putative Binding Site

The family 16 endo-β-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-β-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases.

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