Molecular characterization of the genes encoding the tungsten-containing aldehyde ferredoxin oxidoreductase from Pyrococcus furiosus and formaldehyde ferredoxin oxidoreductase from Thermococcus litoralis

Abstract

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...

Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1

“…Moreover, the complete amino acid sequences of P. furiosus AOR and T. litoralis FOR are 38% identical (59% similar), suggesting that they are similar in structure (23). The AORs and FORs also share amino-terminal homology with the W-containing, aldehyde-oxidizing CAR enzymes of the clostridia (Fig.…”

“…Purified tungstoenzymes include (i) formate dehydrogenase from Clostridium thermoaceticum (50,53) and C. formicoaceticum (12), (ii) carboxylic acid reductase (CAR) from the same two organisms (49,52), (iii) aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (32), (iv) formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis and P. furiosus (19,33), (v) formylmethanofuran dehydrogenase from Methanobacterium wolfei (43) and Methanobacterium thermoautotrophicum (6), and (vi) glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GAPOR) from P. furiosus (34). Amino-terminal amino acid sequence analyses (23,32) indicate strong homology between CAR, FOR, and AOR, suggesting that these enzymes, all of which can utilize aldehydes as substrates, are closely related. On the other hand, formylmethanofuran dehydrogenase and GAPOR (34) show little or no N-terminal homology to the aldehyde-oxidizing enzymes or to each other (data have not been reported for formate dehydrogenase).…”

“…The gene for AOR has been cloned and sequenced (23), and its crystal structure has been determined to a resolution of 2.3 Å (0.23 nm) (10). These data show that the enzyme is a homodimer with a subunit M r of 67,000 and that the subunits are bridged by an iron atom.…”

High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis

“…litoralis is a heterotrophic organism which was initially described as growing on peptides and pyruvate but not on carbohydrates (22). Several enzymes supposedly involved in peptide metabolism in this organism have been described (1,14,17,20,24). In contrast, very little is known about carbohydrate metabolism.…”

“…For maltose transport assays, [U- 14 C]maltose with a specific activity of 629 mCi/mmol (Amersham) was used. For trehalose transport, [U- 14 C]trehalose with a specific activity of 108 mCi/mmol was synthesized as described previously (5). For binding assays, a preparation of 650 mCi/mmol was used.…”