Purification and molecular characterization of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaean Pyrococcus furiosus: The third of a putative five-member tungstoenzyme family

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally n ear 100 degrees C by fermenting peptides and sugars to produce organic acid s, CO2, and H-2. Its growth requires tungsten, and two different tungsten-c ontaining enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldeh yde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously pur ified from P. furiosus, These two enzymes are thought to function in the me tabolism of peptides and carbohydrates, respectively. A third type of tungs ten-containing enzyme, formaldehyde ferredoxin oxidoreductase (POR), has no w been characterized. FOR is a homotetramer with a mass of 280 kDa and cont ains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, togethe r with a pterin cofactor. The low recovery of FOR activity during purificat ion was attributed to loss of sulfide, since the purified enzyme was activa ted up to fivefold by treatment with sulfide (HS-) under reducing condition s. FOR uses P. furiosus ferredoxin as an electron acceptor (K-m = 100 mu M) and oxidizes a range of aldehydes, Formaldehyde (K-m = 15 mM for the sulfi de-activated enzyme) was used in routine assays, but the physiological subs trate is thought to be an aliphatic C-5, semi- Mr dialdehyde, e.g., glutari c dialdehyde (K-m = 1 mM), Based on its amino-terminal sequence, the gene e ncoding FOR (for) was identified In the genomic database, together with tho se 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 addition al land nonlinked genes (termed wor4 and wor5) that encode putative tungsto enzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and W OR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] c luster per subunit.