The crystal structure of the tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus, a hyperthermophilic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal form averaging. AOR consists of two identical subunits, each containing an Fe4S4 cluster and a molybdopterin-based tungsten cofactor that is analogous to the molybdenum cofactor found in a large class of oxotransferases. Whereas the general features of the tungsten coordination in this cofactor were consistent with a previously proposed structure, each AOR subunit unexpectedly contained two molybdopterin molecules that coordinate a tungsten by a total of four sulfur ligands, and the pterin system was modified by an intramolecular cyclization that generated a three-ringed structure. In comparison to other proteins, the hyperthermophilic enzyme AOR has a relatively small solvent-exposed surface area, and a relatively large number of both ion pairs and buried atoms. These properties may contribute to the extreme thermostability of this enzyme.

The archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of carbohydrates to yield acetate, CO2, and H2. Cell-free extracts contain very low activity of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, but extremely high activity of glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). GAPOR was purified under strictly anaerobic conditions. It is a monomeric, O2-sensitive protein of M(r) approximately 63,000 which contains pterin and approximately 1 tungsten and 6 iron atoms per molecule. The enzyme oxidized glyceraldehyde-3-phosphate (Km 28 microM) to 3-phosphoglycerate and reduced P. furiosus ferredoxin (Km 6 microM), but it did not oxidize formaldehyde, acetaldehyde, glyceraldehyde, benzaldehyde, glucose, glucose 6-phosphate, or glyoxylate, nor did it use NAD(P) as an electron acceptor. It is proposed that GAPOR has a glycolytic role and functions in place of glyceraldehyde-3-phosphate dehydrogenase and possibly phosphoglycerate kinase.

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The hyperthermophilic archaea Pyrococcus furiosus and Thermococcus litoralis contain the tungstoenzymes aldehyde ferredoxin oxidoreductase, a homodimer, and formaldehyde ferredoxin oxidoreductase, a homotetramer. Herein we report the cloning and sequencing of the P. furiosus gene aor (605 residues; M r , 66,630) and the T. litoralis gene for (621 residues; M r , 68,941).Enzymes containing tungsten (W) are rare in biology, yet the hyperthermophilic archaea contain three distinct types, all of which catalyze aldehyde oxidation (6-9). The homodimeric aldehyde ferredoxin oxidoreductase (AOR) of Pyrococcus furiosus (maximum growth temperature [T max ], 105ЊC [3]) oxidizes a wide range of aliphatic and aromatic, nonphosphorylated aldehydes (7). Recent crystallographic analysis (refined at 2.3 Å [2]) demonstrated that the two subunits of AOR are bridged by a monomeric iron site and that each subunit contains a [4Fe-4S] cluster and a mononuclear W cofactor. The W site is coordinated by four dithiolene-sulfur ligands from two pterin molecules (2). In contrast to AOR, the homotetrameric formaldehyde ferredoxin oxidoreductase (FOR) of Thermococcus litoralis (T max , 98ЊC [11]) oxidizes only C 1 to C 3 aldehydes, although it also contains one W atom and one [4Fe-4S] cluster per subunit (8). The third W-containing oxidoreductase (GAPOR) is monomeric and specifically oxidizes glyceraldehyde-3-phosphate (9). We have now cloned and sequenced the genes encoding P. furiosus AOR and T. litoralis FOR. The primary structure of a tungstoenzyme has not been previously reported.P. furiosus AOR. For aor, a 7.2-kbp EcoRI P. furiosus (DSM 3638 [3]) genomic fragment was cloned into pBluescript II KS ϩ (pAOR 3-1), using probes derived from the amino-terminal sequence and standard procedures (Southern blotting and hybridization and colony lifts) (12). From pAOR 3-1, 4,951 bp were sequenced, beginning 90 bp upstream of a unique internal EcoRV site and ending at the downstream EcoRI site (Fig. 1). Sequencing was performed with a mixture of deletion clones constructed with appropriate restriction enzymes and by primer walking. The fragment contained the genetic information for the entire AOR polypeptide ( Fig. 1 and 2) together with (i) a P. furiosus gene, termed cmo for cofactor modification, which may code for a cofactor-modifying protein; (ii) the first 169 amino acid residues of a P. furiosus ahc gene encoding an S-adenosylhomocysteine hydrolase; and (iii) a short open reading frame and a partially sequenced long open reading frame of unknown function ( Fig. 1) (5).The gene for AOR contained 605 codons which correspond to a protein with a molecular weight of 66,630 (compared with 85,000 by sodium dodecyl sulfate (SDS)-gel analysis [7]) or 136,066 for the homodimer, including cofactors (Fig. 2). The [4Fe-4S] cluster in each AOR subunit is liganded by the cysteinyl residues at positions 288, 290, 295, and 494 (Fig. 2). The remote Cys-494 residue forms a hydrogen bond to a ring nitrogen of one pterin, thus linking the two metal centers at the...