title: 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.
abstract: Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC 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 uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) 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 C5 semi- 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. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 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.
authors.affiliation: Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: R
authors.initials: R
authors.lastname: Roy
authors.forename: S
authors.initials: S
authors.lastname: Mukund
authors.forename: G J
authors.initials: GJ
authors.lastname: Schut
authors.forename: D M
authors.initials: DM
authors.lastname: Dunn
authors.forename: R
authors.initials: R
authors.lastname: Weiss
authors.forename: M W
authors.initials: MW
authors.lastname: Adams
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D019143:Evolution, Molecular; D005288:Ferredoxins / Q000378:metabolism; D019766:Genes, Archaeal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008667:Metalloproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D020027:Pyrococcus furiosus / Q000201:enzymology / Q000235:genetics*; D019163:Reducing Agents; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013440:Sulfides; D014414:Tungsten / Q000032:analysis*