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Purification, Characterization, and Metabolic Function JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4757–4764 Vol. 177, No. 16 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology Purification, Characterization, and Metabolic Function of Tungsten-Containing Aldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic and Proteolytic Archaeon Thermococcus Strain ES-1 JOHANN HEIDER, KESEN MA, AND MICHAEL W. W. ADAMS* Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602 Received 6 February 1995/Accepted 6 June 1995 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 (S0) for growth, which it reduces to H2S. 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 Mr of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. Electron paramagnetic resonance analyses of the reduced enzyme indicated the presence of a single [4Fe-4S]1 cluster with an S 5 3/2 ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with 21 21 the highest apparent kcat/Km values (>10 mM s ) were acetaldehyde, isovalerylaldehyde, and phenylacet- aldehyde (Km values of <100 mM). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 mM (with crotonaldehyde as the substrate). Thermococcus strain ES-1 AOR also catalyzed the reduction of acetate (apparent Km of 1.8 mM) below pH 6.0 (with reduced methyl viologen as the electron donor) but at much less than 1% of the rate of the oxidative reaction (with benzyl viologen as the electron acceptor at pH 6.0 to 10.0). The properties of Thermococcus strain ES-1 AOR are very similar to those of AOR previously purified from the saccharolytic hyperthermophile Pyrococcus furiosus, in which AOR was proposed to oxidize glyceraldehyde as part of a novel glycolytic pathway (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208–14216, 1991). However, Thermococcus strain ES-1 is not known to metabolize carbohydrates, and glyceraldehyde was a very 21 21 poor substrate (kcat/Km of <0.2 mM s ) 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 glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase in nature (45), a biological requirement for tungsten (W), an (GAPOR) from P. furiosus (34). Amino-terminal amino acid analog of molybdenum (Mo), has only recently been estab- sequence analyses (23, 32) indicate strong homology between lished (4). Indeed, the first naturally occurring W-containing CAR, FOR, and AOR, suggesting that these enzymes, all of enzyme was purified only in the early 1980s (53). Interest in which can utilize aldehydes as substrates, are closely related. tungstoenzymes has greatly intensified in the last few years, On the other hand, formylmethanofuran dehydrogenase and and several different types have been purified (3). So far they GAPOR (34) show little or no N-terminal homology to the have been obtained from methanogenic, acetogenic, and fer- aldehyde-oxidizing enzymes or to each other (data have not mentative anaerobes, all but one of which (Clostridium formi- been reported for formate dehydrogenase). coaceticum) are thermophilic or hyperthermophilic. Very re- The best characterized of the tungstoenzymes at the molec- cent evidence indicates that tungstoenzymes are also present in ular level is AOR from the hyperthermophile P. furiosus (3, 31, mesophilic sulfate-reducing bacteria (18) and in some aerobic 32). The gene for AOR has been cloned and sequenced (23), methylotrophs (16). Purified tungstoenzymes include (i) for- and its crystal structure has been determined to a resolution of mate dehydrogenase from Clostridium thermoaceticum (50, 53) and C. formicoaceticum (12), (ii) carboxylic acid reductase 2.3 Å (0.23 nm) (10). These data show that the enzyme is a M (CAR) from the same two organisms (49, 52), (iii) aldehyde homodimer with a subunit r of 67,000 and that the subunits ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus are bridged by an iron atom. Each subunit also contains a (32), (iv) formaldehyde ferredoxin oxidoreductase (FOR) [4Fe-4S] cluster together with a mononuclear W site coordi- from Thermococcus litoralis and P. furiosus (19, 33), (v) formyl- nated by two molybdopterin molecules. In fact, P. furiosus methanofuran dehydrogenase from Methanobacterium wolfei AOR is the only W-containing, hyperthermophilic, or pterin- (43) and Methanobacterium thermoautotrophicum (6), and (vi) containing enzyme for which a structure is available. However, in spite of the wealth of structural information, the physiolog- ical function of AOR in P. furiosus is far from clear. This * Corresponding author. Mailing address: Department of Biochem- organism grows optimally at 1008C by the fermentation of istry, Life Sciences Bldg., University of Georgia, Athens, GA 30602. carbohydrates (14). We speculated that AOR was part of an Phone: (706) 542-2060. Fax: (706) 542-0229. Electronic mail address: unusual nonphophosphorylated Entner-Doudoroff pathway [email protected]. for sugar catabolism in which it catalyzed the oxidation of 4757 4758 HEIDER ET AL. J. BACTERIOL. glyceraldehyde (32). Although the enzymes of the novel path- TABLE 1. Purification of AOR from ES-1 way were subsequently confirmed by others (41), two recent Amt of Purifi- Activity Sp act Yield nuclear magnetic resonance studies showed that this organism Step protein cation (U) (U/mg) (%) uses an unusual Embden-Meyerhof pathway for the oxidation (mg) (fold) of [13C]glucose (20, 40). The only aldehyde oxidation step in this pathway involves GAP. P. furiosus AOR, however, does Cell extract 12,800 48,100 1.9 1 not utilize GAP as a substrate (32), and GAP oxidation is now Q-Sepharose (pH 8.0) 2,120 111,000 26 100 14 Hydroxyapatite 290 62,700 108 57 57 thought to be catalyzed by another tungsten enzyme in this Superdex pool 123 47,000 191 43 100 organism, GAPOR (34). Q-Sepharose (pH 7.5) 42 20,000 238 18 125 To address the question of the physiological function of Hydroxyapatite 30 13,250 221 12 116 AOR, in this paper we have focused on the novel deep-sea hyperthermophile isolate ES-1 (36), which, by 16S rRNA anal- ysis, has been determined to be a member of the genus Ther- mococcus (27). Thermococcus strain ES-1 grows at tempera- glyceraldehyde as a substrate, the reaction was performed at 458C to prevent the nonenzymatic reduction of benzyl viologen. When methyl viologen was used in tures up to 918C and is an obligately anaerobic heterotroph place of benzyl viologen as the electron acceptor for AOR, its reduction was 0 that reduces S and utilizes proteinaceous materials (peptone, measured at 600 nm by using a molar extinction coefficient of 12,000 M21 cm21 yeast extract, and casein) as a carbon source (36). This organ- (48). The reduction of ES-1 ferredoxin was measured at 400 nm by using an 21 21 ism is not known to metabolize sugars (27, 36), and yet cell extinction coefficient of 7,000 M cm for the difference between the oxidized and reduced forms of the protein. The purification and properties of ES-1 extracts contain high aldehyde oxidation activity as measured ferredoxin will be described elsewhere (54). The ability of AOR to catalyze the with crotonaldehyde as the substrate (27), the usual assay for reduction of acetate was determined at 858C in 500 mM potassium phosphate P. furiosus AOR activity (32). It was therefore of great interest (pH 5.5). The electron donor was methyl viologen (10 mM) which was completely to characterize the enzyme responsible for catalyzing this ac- reduced by sodium dithionite. After the enzyme was added and the mixture was preincubated for 1 min, the reaction was initiated by adding sodium acetate (final tivity in Thermococcus strain ES-1, to assess its relationship to concentration, 60 mM) and was monitored by measuring the oxidation of re- P. furiosus AOR, and to investigate its potential physiological duced methyl viologen at 600 nm. roles by kinetic and substrate analyses. The molecular weight of AOR was estimated by gel filtration on a column of Superdex 200 (1.6 by 60 cm; Pharmacia-LKB) and by nondenaturing gel elec- trophoresis using polyacrylamide concentrations (wt/vol) of 4, 5, 6, 7, 7.5, and MATERIALS AND METHODS 8%. For both methods, catalase (240,000), lactate dehydrogenase (140,000), yeast alcohol dehydrogenase (150,000), bovine serum albumin (67,000), and egg Growth of organism and enzyme purification. Thermococcus strain ES-1 albumin (45,000) were used as standard proteins and the data were analyzed by (hereafter referred to as ES-1) was obtained from John Baross of the University standard methods (13). SDS-polyacrylamide gel electrophoresis was performed of Washington (36). It was grown in a 600-liter fermentor under anaerobic on 10% (wt/vol) polyacrylamide gels by the method of Laemmli (24). SDS conditions at 818C with S0 at a final concentration of 0.04 g/liter as previously molecular weight markers were purchased from Sigma Chemical Co. (St. Louis, described (27). AOR was purified under anaerobic conditions (9) from 500 g Mo.). Protein concentrations were routinely estimated by the method of Brad- (wet weight) of cells at 258C. Frozen cells were lysed, and a cell extract was ford (8) with bovine serum albumin as the standard.
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