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

Abstract

Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91؇C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S 0 ) for growth, which it reduces to H 2 S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M r of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. ) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.Although molybdenum-containing enzymes are ubiquitous in nature (45), a biological requirement for tungsten (W), an analog of molybdenum (Mo), has only recently been established (4). Indeed, the first naturally occurring W-containing enzyme was purified only in the early 1980s (53). Interest in tungstoenzymes has greatly intensified in the last few years, and several different types have been purified (3). So far they have been obtained from methanogenic, acetogenic, and fermentative anaerobes, all but one of which (Clostridium formicoaceticum) are thermophilic or hyperthermophilic. Very recent evidence indicates that tungstoenzymes are also present in mesophilic sulfate-reducing bacteria (18) and in some aerobic methylotrophs (16). 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 best characterized of the tungstoenzymes at the molecular level is AOR from the hyperthermophile P. furiosus (3,31,32). 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 wit...

Single gene insertion drives bioalcohol production by a thermophilic archaeon

“…It is thought that the in vivo function of AOR is to oxidize toxic aldehydes generated from the 2-ketoacids that are produced during sugar and peptide fermentation. However, this hypothesis has not been experimentally verified (20).…”

“…It is not clear if AOR normally generates any acetaldehyde from acetate in wild-type P. furiosus (lacking AdhA) and, if so, how that the acetaldehyde is further metabolized. In any event, the properties of strain A call into question the previously proposed role of AOR (20).…”

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

“…We therefore propose (Fig. 4) that under S 0 -sufficient conditions, the aldehydes produced from 2-keto acids are oxidized by AOR with the production of reduced ferredoxin (28) but that under conditions of S 0 limitation, these aldehydes are reduced by ADH. Reduction of aldehydes by ADH rather than their oxidation by AOR generates an oxidized electron carrier (NADP) rather than a reduced one (reduced ferredoxin).…”

“…These enzymes might function in the reverse direction to produce aldehydes with concomitant oxidation of reduced ferredoxin. However, ES-1 AOR was recently purified, and from kinetic analyses it was concluded that the reduction of organic acids is unlikely to be the physiological reaction (28). Moreover, the most efficient aldehyde substrates for ES-1 AOR were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde, which are derivatives of the substrates for the keto acid oxidoreductases found in ES-1 (Table 1).…”

Glyceraldehyde-3-Phosphate Ferredoxin Oxidoreductase fromMethanococcus maripaludis

“…In contrast, the GAPOR activity of crude lysates of M. maripaludis was irreversibly inactivated by exposure to air. Most other previously characterized nonrecombinant AOR proteins (including GAPOR Pf ) also appear to be irreversibly inactivated by oxygen exposure (16,26,28). The response of recombinant GAPOR Mm to L-cysteine reactivation may be related to the sulfide activation observed for a Wcontaining formaldehyde oxidoreductase purified from P. furiosus (34).…”