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Comparative Study
.1999 Feb;181(4):1171-80.
doi: 10.1128/JB.181.4.1171-1180.1999.

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

Affiliations
Comparative Study

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

R Roy et al. J Bacteriol.1999 Feb.

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.

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Figures

FIG. 1
FIG. 1
SDS gel electrophoresis analysis of purifiedP. furiosus FOR. Samples of FOR (8.5 mg/ml) were incubated with an equal volume of SDS (1%, wt/vol) at 100°C for 10 min (lane 3) or 30 min (lane 2) prior to electrophoresis on a 10% (wt/vol) acrylamide gel. Lane 1 contained marker proteins with the indicated molecular masses (in kilodaltons) for (from top to bottom) myosin, β-galactosidase, phosphorylaseb, bovine serum albumin, ovalbumin, and carbonic anhydrase.
FIG. 2
FIG. 2
N-terminal amino acid sequence analysis ofP. furiosus FOR and related enzymes. Abbreviations: Pf FOR,P. furiosus FOR (this work); ES AOR, ES-1 AOR (21); Pf AOR,P. furiosus AOR (36); Tl FOR,T. litoralis FOR (37); Cf CAR,Clostridium formicoaceticum carboxylic acid reductase (49); Pf GAP,P. furiosus GAPOR (38); Ct CAR,Clostridium thermoaceticum carboxylic acid reductase (α-subunit) (47); HVOR,P. vulgaris HVOR (48). Identical amino acids are in boldface type and underlined.
FIG. 3
FIG. 3
Activation ofP. furiosus FOR by sulfide. The enzyme (10 mg/ml in 50 mM Tris-HCl, pH 8.0) was incubated at 23°C with either sodium sulfide (20 mM, open squares), sodium dithionite (20 mM, filled squares), or both (filled circles). At the indicated times, samples were removed and the residual activities were determined with formaldehyde as the substrate under standard assay conditions.
FIG. 4
FIG. 4
Alignment of the sequences of FOR, AOR, and GAPOR and the putative gene products WOR4 and WOR5 fromP. furiosus. Identical residues are boxed, and similar residues are shaded.
FIG. 5
FIG. 5
Phylogenetic relation of the amino acid sequences of tungstoenzymes fromP. furiosus and their homologs found in genome databases. Sequences demarcated by brackets and Roman numerals indicate groups. Asterisks denote amino acid sequence homologous to a tungstoenzyme of known function, though the relevant enzyme activity has not yet been determined in these organisms. Length of the line represents distance in arbitrary units. Abbreviations: Ph,P. horikoshii; Pf,P. furiosus; Mj,M. jannaschii; Pa,Pyrobaculum aerophilum; Af,A. fulgidus. The GenBank accession numbers of amino acid sequences used in this figure are as follows: Ph GAPOR*, g3130733; Mj GAPOR, U67559; Ph WOR5, g3131172; Ph WOR6, g3131173; Ph WOR4, g3130250; Ph FOR*, g3257694; Ph AOR*, g3131315; Af AOR*, g2648240; Af WOR3, g2650295; Af WOR4, g2650628; and Af WOR2, g2650571. See the text for accession numbers of theP. furiosus sequences. For Pa FOR*, Pa AOR*, and Pa WOR3 accession numbers, see reference .
FIG. 6
FIG. 6
Alignments of the cofactor-binding motifs of FOR, AOR, and GAPOR and of the putative gene products WOR4 and WOR5 fromP. furiosus. The numbers in parentheses indicate the numbers of residues between the indicated motifs. See the text for details.
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