Aldehyde ferredoxin oxidoreductase from the hyperthermophilic archaebacterium Pyrococcus furiosus contains a tungsten oxo-thiolate center

1992 ◽  
Vol 114 (9) ◽  
pp. 3521-3523 ◽  
Author(s):  
Graham N. George ◽  
Roger C. Prince ◽  
Swarnalatha Mukund ◽  
Michael W. W. Adams
Keyword(s):  
Pyrococcus Furiosus ◽  
Ferredoxin Oxidoreductase ◽  
Aldehyde Ferredoxin Oxidoreductase

Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

1999 ◽  
Vol 181 (4) ◽  
pp. 1171-1180 ◽  
Author(s):  
Roopali Roy ◽  
Swarnalatha Mukund ◽  
Gerrit J. Schut ◽  
Dianne M. Dunn ◽  
Robert Weiss ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Sequence Similarity ◽  
Sequence Motif ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon ◽  
Ferredoxin Oxidoreductase ◽  
Amino Terminal ◽  
Reducing Conditions ◽  
Aldehyde Ferredoxin Oxidoreductase ◽  
Formaldehyde Ferredoxin Oxidoreductase

ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS−) under reducing conditions. FOR usesP. furiosus ferredoxin as an electron acceptor (Km = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosuschromosome. Two additional (and nonlinked) genes (termedwor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.

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.

Aldehyde Ferredoxin Oxidoreductase

2011 ◽  
Author(s):  
Roopali Roy ◽  
Ish K Dhawan ◽  
Michael K Johnson ◽  
Douglas C Rees ◽  
Michael WW Adams
Keyword(s):  
Ferredoxin Oxidoreductase ◽  
Aldehyde Ferredoxin Oxidoreductase

scholarly journals The thermophilic biomass-degrading bacterium Caldicellulosiruptor bescii utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis

2019 ◽  
Vol 294 (25) ◽  
pp. 9995-10005 ◽  
Author(s):  
Israel M. Scott ◽  
Gabriel M. Rubinstein ◽  
Farris L. Poole ◽  
Gina L. Lipscomb ◽  
Gerrit J. Schut ◽  
...  
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Lag Phase ◽  
Cellulolytic Bacterium ◽  
Ferredoxin Oxidoreductase ◽  
Caldicellulosiruptor Bescii ◽  
Degrading Bacterium ◽  
Growth Optimum ◽  
Cell Densities ◽  
Aldehyde Ferredoxin Oxidoreductase

Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C5 and C6 sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H2 concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

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