Purification, Characterization, and Metabolic Function

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JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4757–4764 Vol. 177, No. 16 0021-9193/95/$04.00ϩ0 Copyright ᭧ 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]؉ cluster ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with 2/3 ؍ with an S ؊1 ؊1 the highest apparent kcat/Km values (>10 ␮M s ) were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde (Km values of <100 ␮M). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 ␮M (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 ؊1 ؊1 poor substrate (kcat/Km of <0.2 ␮M 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 physiological function of AOR in P. furiosus is far from clear. This * Corresponding author. Mailing address: Department of Biochem- organism grows optimally at 100ЊC 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 analysis, 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 45ЊC to prevent the nonenzymatic reduction of benzyl viologen. When methyl viologen was used in tures up to 91ЊC 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 MϪ1 cmϪ1 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 Ϫ1 Ϫ1 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 85ЊC 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 ␮M) 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 electrophoresis 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 81ЊC 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 25ЊC. Frozen cells were lysed, and a cell extract was ford (8) with bovine serum albumin as the standard. A complete metal analysis prepared as described previously (27). This extract was directly loaded on a (40 elements, including tungsten and iron) was performed by plasma emission column (8 by 15 cm) of Q-Sepharose Fast Flow (Pharmacia-LKB) equilibrated spectroscopy using a Jarrel Ash Plasma Comp 750 instrument at the Center for with buffer A (50 mM Tris-HCl [pH 8.0] containing 10% [vol/vol] glycerol, 2 mM Complex Carbohydrate Research, University of Georgia. The iron and acid- dithiothreitol, and 2 mM sodium dithionite). The column was eluted with a labile sulfide contents of pure AOR were measured by using o-phenanthroline 10-liter linear 0 to 0.5 M NaCl gradient in buffer A. The flow rate was 15 ml/min, (26) and by methylene blue formation (11). The N-terminal sequence of AOR and 150-ml fractions were collected. AOR activity started to elute from the was determined with an Applied BioSystems model 477 sequencer (13). Apo- column as 0.20 M KCl was applied. Fractions containing AOR activity were protein was prepared and reduced with dithiothreitol under anaerobic conditions combined in three separate pools (1,600 ml total), and each pool was separately by previously described methods (5). The cysteine content of the reduced ap- loaded on a column (5 by 12 cm) of hydroxyapatite (American International oprotein was estimated from the apoprotein’s reaction with 5,5Ј-dithiobis-(2- Chemical) equilibrated with buffer A. Each column was eluted at a flow rate of nitrobenzoic acid) (38). Pterin derivatives were extracted from acid-treated AOR 3 ml/min with a 1.2-liter linear 0 to 0.20 M potassium phosphate (pH 8.0) with and without iodine according to the procedure of Yamamoto et al. (53). gradient in buffer A, and fractions of 100 ml were collected. AOR activity started Cross-linking of AOR with dimethyl suberimidate was carried out as described to elute as 60 mM phosphate was applied. Fractions containing AOR activity by Kessler et al. (22). Electron paramagnetic resonance (EPR) spectra were from all three columns were pooled (600 ml) and concentrated by ultrafiltration recorded on an IBM-Bruker ER 300D spectrometer interfaced to an ESP 3220 with an Amicon type PM-30 membrane. The concentrated fraction (20 ml) was Data System and equipped with an Oxford Instruments ITC-4 flow cryostat. applied to a column (6 by 60 cm) of Superdex 200 (Pharmacia-LKB) equilibrated with buffer A containing 200 mM NaCl. The flow rate was 2 ml/min, and fractions RESULTS of 10 ml were collected. Those fractions with a specific activity for AOR above 150 U/mg were combined (30 ml) and applied to a Q-Sepharose (High-Perfor- mance) column (1.6 by 10 cm; Pharmacia-LKB) equilibrated with buffer B (50 Purification of AOR. Cell extracts of ES-1 contained signif- mM Tris-HCl [pH 7.5] containing 10% [vol/vol] glycerol, 2 mM dithiothreitol, icant aldehyde oxidoreductase activity using crotonaldehyde as and 2 mM sodium dithionite). The column was eluted with a 600-ml linear 0 to the substrate and benzyl viologen as the electron carrier, with 0.30 M NaCl gradient at 3 ml/min, and 10-ml fractions were collected. AOR specific activities ranging from 1.9 to 4.5 U/mg (from three activity began to elute as 130 mM NaCl was applied. Those fractions containing AOR activity above 210 U/mg were combined (50 ml) and were applied to a different cell batches). With ES-1, more than 95% of the ac- hydroxyapatite column (2.6 by 25 cm; Bio-Rad) equilibrated with buffer B. The tivity remained in the supernatant after centrifugation of the column was eluted with a 600-ml linear 0 to 0.10 M potassium phosphate (pH cell extract (50,000 ϫ g for 80 min at 4ЊC), indicating that AOR 7.5) gradient in buffer B at 1 ml/min, and 25-ml fractions were collected. AOR is a cytoplasmic enzyme. The time required for a 50% loss of eluted as 50 mM phosphate was applied. Those fractions containing pure AOR as judged by sodium dodecyl sulfate (SDS) electrophoresis were combined (50 activity upon exposure of the extract to air was less than 2 min; ml), concentrated by ultrafiltration to 5 ml, and stored frozen at Ϫ80ЊC. hence, the enzyme was purified under strictly anaerobic and Other methods. AOR activity was routinely determined by measuring the reducing conditions. Results of a typical purification are sum- crotonaldehyde-dependent reduction of benzyl viologen (1.6 mM) in 100 mM marized in Table 1, and an SDS-gel analysis of the respective EPPS buffer (pH 8.4) at 85ЊC in serum-stoppered cuvettes under anaerobic peak fractions is shown in Fig. 1A. The enzyme was purified conditions (32). To remove traces of O2, sodium dithionite was added to the assay mixture (at 85ЊC) to give an A600 of about 0.2. The enzyme was then added, about 120-fold, with a recovery of activity of about 12%. AOR and the reaction was started after a 2-min incubation by adding crotonaldehyde therefore appears to constitute ca. 1% of the total protein to a final concentration of 400 ␮M. The molar extinction coefficient at 600 nm for content of ES-1. reduced benzyl viologen was determined to be 7,400 MϪ1 cmϪ1. The same assay conditions were used to examine other substrates for AOR, and in all cases Molecular properties of AOR. Purified AOR gave rise to a activities are expressed as micromoles of substrate oxidized per minute. With single protein band after SDS-gel electrophoresis (Fig. 1A), VOL. 177, 1995 TUNGSTEN-CONTAINING ALDEHYDE FERREDOXIN OXIDOREDUCTASE 4759

FIG. 1. (A) SDS-gel analysis of ES-1 AOR during puriﬁcation. The samples were as follows: lanes 1 and 8, markers; lane 2, cell extract; lane 3, Q-Sepharose (pH 8.0) fractions; lane 4, hydroxyapatite fractions; lane 5, Superdex 200 fractions; lane 6, Q-Sepharose (pH 7.5) fractions; and lane 7, hydroxyapatite fractions. Each lane contained 10 ␮g of protein, and a 10% (wt/vol) acrylamide gel was used. (B) Cross-linking of ES-1 AOR with dimethyl suberimidate. AOR (2.0 mg/ml in 200 mM triethanolamine buffer [pH 8.5] containing 4 mM dithiothreitol and 0.4 mM sodium dithionite) was incubated with 7 mM dimethyl suberimidate (freshly made in the same buffer), and samples were removed for SDS-gel analysis after 1 min (lane 3), 30 min (lane 4), 150 min (lane 5), 280 min (lane 6), and 400 min (lane 7). Lane 1 contained markers, and lanes 2 and 8 contained untreated AOR. A 10% (wt/vol) acrylamide gel was used.

and this band corresponded to an Mr of 75,000 Ϯ 5,000. The and 0.2 g-atom per subunit, respectively. The amounts of iron presence of a single type of subunit in AOR was confirmed by and tungsten in the enzyme were confirmed by colorimetric amino-terminal sequence analysis. This analysis gave rise to a analyses, which gave values of 4.5 Ϯ 0.5 g-atom of Fe and 1.0 single sequence which showed a high level of homology to P. Ϯ 0.2 g-atom of W. The acid-labile sulfide content of AOR was furiosus AOR as well as to FOR from T. litoralis and CAR from 5.9 Ϯ 2.0 g-atom per subunit, and the number of cysteinyl clostridia, but not to P. furiosus GAPOR (Fig. 2). The apparent residues was 7.0 Ϯ 2.0 per subunit. The presence of a pterin

Mr of the native AOR was estimated to be 105,000 Ϯ 10,000 cofactor in AOR was demonstrated by extracting it from the after gel filtration on Superdex S-200, and the same result was enzyme in the presence and absence of iodine and measuring obtained by a Ferguson plot analysis of the enzyme’s migration the fluorescence spectra of the resulting derivatives (Fig. 3). on nondenaturing gels (data not shown). After treatment of Similar spectra have been reported for P. furiosus AOR (32) the enzyme with the cross-linking reagent dimethyl suberimi- and T. litoralis FOR (33), as well as for all molybdenum- date, the apparent Mr of the protein after SDS-gel analysis containing enzymes (with the exception of nitrogenase) when increased to approximately 100,000 (Fig. 1B). These results they were treated in a similar fashion (37). The spectra shown suggest that native AOR is a homodimer which exhibits anom- in Fig. 3 correspond to the form A (prepared in the presence alous behavior both in the native state and after treatment with of iodine) and form B (no iodine) derivatives of molybdopterin SDS. This conclusion is supported by the properties of P. (37). furiosus AOR. This enzyme gives results virtually identical to Catalytic properties of AOR. The enzyme was purified by those of ES-1 AOR when analyzed by gel filtration and SDS- monitoring its ability to catalyze the crotonaldehyde-depen- gel electrophoresis (31, 32), and yet from the DNA sequence of its gene (23) and its X-ray structure (10), P. furiosus AOR is concluded to be a homodimer with a subunit Mr of 67,000. Hence, the calculations presented herein are based on the assumption that ES-1 AOR is a dimeric protein with an Mr of 67,000 per subunit. An elemental analysis of ES-1 AOR by inductively coupled plasma emission spectroscopy revealed the presence (per subunit) of 3.6 Ϯ 1.0 g-atom of Fe, 0.9 Ϯ 0.1 g-atom of W, 1.1 Ϯ 0.1 g-atom of Mg, and 2.0 Ϯ 0.2 g-atom of P. The only other metals detected in significant amounts (Ͼ0.1 g-atom per subunit) were Ca and Zn, which were present at approximately 0.7

FIG. 2. Amino-terminal amino acid sequences of ES-1 AOR and related enzymes. The abbreviations used and sources of the data are as follows: ES, ES-1 (this work); Pf AOR, P. furiosus AOR (32); T1 FOR, T. litoralis FOR (33); FIG. 3. Fluorescence spectra of material from denatured ES-1 AOR pre- Cf CAR, C. formicoaceticum CAR (52); Ct CAR, C. thermoaceticum CAR pared in the presence (Aex and Aem) and absence (Bex and Bem) of iodine. The (64,000-Mr ␣-subunit) (46); Pf GAPOR, P. furiosus GAPOR (34). Gaps have emission spectra (Aem and Bem) were recorded at 400 nm (excitation wave- been inserted in the Cf CAR sequence to maximize identity with ES-1 AOR. length), and the excitation spectra (Aex and Bex) were recorded at 470 nm Identical residues are in bold. (emission wavelength). 4760 HEIDER ET AL. J. BACTERIOL.

FIG. 5. Dependence of the activity of ES-1 AOR on temperature. Enzyme activity was measured by the crotonaldehyde-dependent reduction of benzyl viologen under standard assay conditions except that the temperature was varied as indicated. The inset shows the corresponding Arrhenius plot. sp. act, speciﬁc activity; T, temperature.

aromatic aldehydes as substrates, and the associated kinetic parameters are summarized in Table 2. All values were ob- FIG. 4. Dependence of AOR activity on pH and electron carrier. (A) AOR tained from Lineweaver-Burk plots (data not shown) which activity was measured at 80ЊC by crotonaldehyde oxidation with benzyl viologen were linear over extended concentration ranges (except for the (open squares and closed triangles) or methyl viologen (closed circles) as the substrate inhibition noted below). Those with the highest ap- electron carrier. All buffers were at a ﬁnal concentration of 100 mM, and the pH was determined at 85ЊC. The buffers used were as follows: open squares, potas- parent kcat/Km values corresponded to the aldehyde derivatives sium phosphate (pH 4.7 to 11.0); solid triangles, sodium acetate (pH 4.2 and 4.7), of amino acids with aromatic, branched, and short to medium 2-(N-morpholino)ethanesulfonic acid (MES) (pH 4.9 and 5.2), piperazine-N,NЈ- aliphatic side groups. All exhibited apparent Km values of less bis-(2-ethanesulfonic acid) (PIPES) (pH 6.4), 3-(N-morpholino)propanesulfonic than 100 ␮M with benzyl viologen as the electron acceptor at acid (MOPS) (pH 6.8), N-(2-hydroxyethyl)-piperazine-NЈ(2-ethanesulfonic acid) (HEPES) (pH 7.2), N-(2-hydroxyethyl)piperazine-NЈ-3-propanesulfonic acid a concentration of approximately 10 times its apparent Km (EPPS) (pH 7.9 and 8.2), 2-(N-cyclohexylamino)-ethanesulfonic acid (CHES) value (Table 2). The apparent Km value for ES-1 ferredoxin (pH 8.7), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) (pH 9.6); was very low (10 ␮M, with crotonaldehyde as the substrate), solid circles, pH 4.9 and 5.3, MES (pH 4.9 and 5.3), PIPES (pH 6.4), MOPS (pH which is consistent with its being the physiological electron 6.8), HEPES (pH 7.2), EPPS (pH 7.9 and 8.2), CHES (pH 9.0), CAPS (pH 9.6 and 10.6), and potassium phosphate (pH 11.0 and 12.0). (B) AOR activity was carrier for the enzyme. Interestingly, the apparent Km for cro- measured at 85ЊC by the reduction of acetate in 500 mM potassium phosphate tonaldehyde was unaffected by lowering the assay temperature buffer at the indicated pH. Methyl viologen (10 ␮M) reduced by a slight excess from 85 to 45ЊC, even though this resulted in an order of of sodium dithionite was the electron donor. The reaction was initiated by the addition of sodium acetate (60 mM).

TABLE 2. Kinetic parameters of ES-1 AOR

Substrate or Apparent Km Ϫ1 kcat/Km dent reduction of benzyl viologen. As shown in Fig. 4, AOR a kcat (s ) Ϫ1 Ϫ1 was active over a broad pH range with a general increase in electron carrier (␮M) (␮M s ) activity with increasing pH with two different buffer systems. Acetaldehyde 16 343 22 Figure 4 also shows that the enzyme was much less active Benzaldehyde 57 720 13 throughout the pH range when benzyl viologen was replaced Phenylacetaldehyde 76 960 13 by methyl viologen. It should be noted that activities measured Isovalerylaldehyde 28 272 10 at pH values of 11 and above are estimates because under Propionaldehyde 150 1,100 7.5 these conditions the nonenzymatic reduction of the viologen Crotonaldehyde 136 269 2.0 Indoleacetaldehyde 50 55 1.1 dye was signiﬁcant. Presumably, the more hydrophobic nature Formaldehyde 1,422 950 0.7 and higher reduction potential of benzyl viologen (E0ЈϭϪ360 Salicylaldehyde 65 13 0.2 b mV) compared with methyl viologen (E0ЈϭϪ440 mV) facil- Crotonaldehyde 130 22 0.17 itates the AOR reaction (see below). The temperature depen- Glyceraldehydeb 196 2.8 0.015 dence of AOR activity is shown in Fig. 5. The optimum was ES-1 Ferredoxin 10 270 27 85ЊC, and a linear Arrhenius plot was obtained by using data Benzyl viologen 169 250 1.5 up to this temperature. An activation energy of 75 kJ/mol was Methyl viologen 2,900 62 0.011 c calculated. That AOR was not particularly thermostable was Acetate 1,800 1.6 0.001 conﬁrmed by results from prolonged incubation studies at high a Except where indicated, reactions were carried out at 85ЊC in 100 mM EPPS temperatures. When the pure enzyme was used at a concen- [N-(2-hydroxyethyl)piperazine-NЈ-3-propanesulfonic acid] buffer (pH 8.4) with tration of 2.0 mg/ml, the times required for a 50% loss of benzyl viologen (1.6 mM) as the electron carrier or crotonaldehyde (1.0 mM) as catalytic activity (when assayed at 85ЊC) at 70 and 85ЊC were 5 the substrate. b Assayed at 45ЊC. and 1 min, respectively. c Using reduced methyl viologen (10 ␮M) as the electron donor in 500 mM ES-1 AOR was able to utilize a range of both aliphatic and potassium phosphate buffer (pH 5.5). VOL. 177, 1995 TUNGSTEN-CONTAINING ALDEHYDE FERREDOXIN OXIDOREDUCTASE 4761 magnitude decrease in activity (Table 2). ES-1 AOR was un- because of the inability of sodium dithionite, which is unstable able to reduce NAD or NADP (with crotonaldehyde as the in acidic media (30), to reduce methyl viologen. There was no substrate) or to oxidize glucose, GAP, glyoxylate, or succinyl- reaction when methyl viologen was replaced by benzyl violo- semialdehyde (each up to a 10 mM concentration with benzyl gen, which also suggests that acid reduction is strongly depen- viologen as the electron carrier). dent upon the overall reduction potential. With reduced

Only a very low apparent kcat/Km value was determined for methyl viologen (10 ␮M) as the electron donor, a linear Line- glyceraldehyde as a substrate for ES-1 AOR. This aldehyde weaver-Burk plot was obtained by using acetate concentrations was previously speculated to be the physiological substrate for over the range 0.12 to 60 mM, and from this plot the apparent

P. furiosus AOR in an unusual glycolytic pathway (32). How- Km for acetate was estimated to be 1.8 mM. The highest acid ever, the activity of AOR with glyceraldehyde as a substrate reduction activity observed (at pH 5.5 with 60 mM acetate as had to be measured at 45ЊC because glyceraldehyde is oxidized the substrate) was approximately 5% of the aldehyde oxidation nonenzymatically at a very high rate at temperatures above activity (with 1 mM crotonaldehyde as the substrate and 5 mM this, as shown by the reduction of the viologen dye (32). Nev- methyl viologen as the electron acceptor) measured under the ertheless, by using the effect of temperature on crotonaldehyde same conditions (Fig. 4). However, this comparison is probably oxidation activity as a guide and assuming that the apparent Km not meaningful, as the concentration of reduced viologen used for glyceraldehyde does not decrease with increasing temper- in the acid reduction assay (10 ␮M) was likely significantly ature, the apparent kcat/Km value for glyceraldehyde at 85ЊC below the Km value. For unknown reasons, no acid reduction can be estimated to be less than 0.2 ␮MϪ1 ⅐ sϪ1. This is an activity was detected when the reduced viologen was increased order of magnitude less than that observed with the aldehydes to 200 ␮M. derived from amino acids described above. The low activity Inhibitors of AOR. Pure AOR was extremely sensitive to with glyceraldehyde compared with, for example, propionalde- inactivation by O2. When the pure enzyme (2.0 mg/ml in 50 hyde (Table 2) may be attributable to glyceraldehyde’s addi- mM Tris-HCl [pH 8.0] containing 10% [vol/vol] glycerol, 2 mM tional hydroxyl groups. This notion is supported by the equally dithiothreitol, and 2 mM sodium dithionite) was briefly shaken low reaction rate with salicylaldehyde compared with benzal- in air to oxidize the dithionite and then left exposed to air at dehyde (Table 2), from which it differs by a single hydroxyl 23ЊC, 50% of the activity was lost within 1 min. No activity was group. Notably, the position of the hydroxyl group relative to regained if the same sample was then degassed with Ar and the carbonyl group is the same in salicylaldehyde and glycer- reduced by the addition of sodium dithionite. Curiously, the aldehyde. In any event, ES-1 AOR is similar to P. furiosus extent of inactivation of the enzyme by O2 greatly depended on AOR in the substrates that it utilizes, and it is clearly distinct the temperature of incubation. For example, at 60, 40, 25, and from T. litoralis FOR, which oxidizes only short-chain aliphatic 4ЊC, the residual activities remaining after a 1-h exposure to air aldehydes (33). were 3, 8, 19, and 39%, respectively, and there was no dramatic ES-1 AOR was inhibited by high concentrations of the alde- change in these activities even after a further1hinair(except hydes that were used as substrates, and different ones affected for the 4ЊC sample, in which the activity decreased to 23% of the enzyme to different degrees. For example, the concentra- the original value). The reason for the substantial residual tions required to produce 50% inhibition for acetaldehyde, activity even after prolonged exposure of the enzyme to O2 is isovalerylaldehyde, and phenylacetaldehyde were approxi- not known. mately 5, 4, and 7 mM, respectively, whereas for crotonalde- Arsenite and iodoacetate are known to inhibit various mo- hyde and propionaldehyde the values were 20 and 0.6 mM, lybdopterin-containing enzymes, including P. furiosus AOR respectively. Thus, the inhibitory concentrations of the sub- and T. litoralis FOR (32, 33). The effects of these compounds strates tested were not correlated with their apparent Km val- on ES-1 AOR were tested by incubating each of them (at a ues (Table 2). For these substrates and the others listed in final concentration of 5 mM) with the enzyme at 23ЊC (in 50 Table 2, the determination of kinetic parameters was not af- mM Tris-HCl [pH 8.0]), and samples were periodically re- fected by substrate inhibition. This was not the case with phe- moved and assayed under standard conditions. With iodoac- nylpropionaldehyde, for which substrate inhibition was appar- etate and arsenite, 50% of the initial activity was lost after 5 ent at a concentration of 150 ␮M. The kinetics of the inhibition and 40 min, respectively. These results are comparable to those were not analyzed. Substrate inhibition also appeared not to be reported for P. furiosus AOR (32), which is much more sensi- related to the product of the reaction, as AOR activity with tive than T. litoralis FOR (33). In addition, ES-1 AOR was acetaldehyde or phenylacetaldehyde as a substrate was unaf- inhibited by approximately 50% when each of the following fected when a high concentration of the corresponding organic reagents was included in the standard assay mixture: p-chloro- acid (250 mM acetate or 100 mM phenylacetate) was also mercuribenzoate (10 ␮M), ZnCl2 (0.25 mM), iodoacetate (0.5 present in the assay mixture. Like ES-1 AOR, P. furiosus AOR mM), sodium arsenite (0.5 mM), and 2,2Ј-bipyridyl (10 mM). was reported to be inhibited by aldehyde substrates (32), and However, 2,2Ј-bipyridyl (10 mM) and potassium cyanide (5 this contrasts with the behavior of FOR from T. litoralis (33). mM) had no significant inhibitory effect when they were incu- The activity of ES-1 AOR in the reverse direction, the re- bated with the enzyme for 2 h prior to assay in the absence of duction of an organic acid, was also investigated. As shown in the reagent (Ͼ90% of the activity remained). The close simi- Fig. 4, the enzyme catalyzed the acetate-dependent oxidation larity in structure between 2,2Ј-bipyridyl and benzyl viologen of reduced methyl viologen but it did so at a significant rate suggests that the former may inhibit during the assay as a only at low pH. There was no reaction in the absence of substrate analog rather than as a chelator. The lack of a sig- enzyme or of acetate. At pH 5.5, the reaction ceased when nificant effect with cyanide is not very surprising, as although about 50% of the viologen had been oxidized, but it continued this compound inactivates some other pterin enzymes (33), the upon addition of sodium dithionite to regenerate the fully AOR of P. furiosus was also relatively insensitive (10% inhibi- reduced form. This cycle could be repeated several times, sug- tion after 1 h with 8 mM cyanide). gesting that the increase in the reduction potential caused by EPR properties of AOR. The EPR spectrum of dithionite- the accumulation of oxidized methyl viologen was inhibitory, reduced (pure) ES-1 AOR recorded near5Kisshown in Fig. rather than the accumulation of the other reaction product, 6. This spectrum is dominated by resonances at g ϭ 4.7, 3.4, acetaldehyde. Assays could not be carried out below pH 5.5 and 1.9 which can be reasonably assigned to the ground state 4762 HEIDER ET AL. J. BACTERIOL.

onances seen with the ES-1 enzyme arise from the coupling of the reduced cluster to a second paramagnetic center, which is thought to be the tungstopterin site (10). The reduction potential of this center in ES-1 AOR appears to be lower than that of the corresponding center in the P. furiosus enzyme (31). The significance of this situation in catalytic terms, if any, is unknown. In spite of the difference in substrate specificity, the AORs of ES-1 and P. furiosus are clearly related to the FORs of P. furiosus and T. litoralis, as both types catalyze aldehyde oxidation, each contains one [4Fe-4S] cluster and one mononuclear tungstopterin site per subunit, and they have amino-terminal sequence homology (19). 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-termi- FIG. 6. EPR spectrum of dithionite-reduced ES-1 AOR. The enzyme (19.8 nal homology with the W-containing, aldehyde-oxidizing CAR mg/ml) was prepared in 50 mM Tris-HCl buffer (pH 7.5) containing 10% (vol/ enzymes of the clostridia (Fig. 2) (49, 52). CARs were discov- vol) glycerol, 2 mM dithiothreitol, and 2 mM sodium dithionite. The spectrometer settings were as follows: gain, 1.6 ϫ 105; modulation amplitude, 0.62 mT; ered by their ability to reduce carboxylic acids to the corre- time constant, 41 ms; sweep time, 670 s; and microwave frequency, 9.56 GHz. sponding aldehyde (49, 52). Substrates include both aliphatic The spectrum was recorded at 5.3 K by using 10 mW of microwave power. and aromatic acids with apparent Km values in the range of 1 to 50 mM (49). Using reduced viologen as the electron donor, the enzymes of C. formicoaceticum and C. thermoaceticum cat- doublet of an S ϭ 3/2 center (with positive axial zero field alyze the reduction of aliphatic carboxylic acids (at pH 6.0) at splitting and significant rhombic distortion) (see the discussion 5 to 10% of the rate at which they oxidize the corresponding in reference 31). These resonances are reminiscent of those aldehyde (at pH 9.0) (49, 52). These results are similar to those seen with P. furiosus AOR when poised in an intermediate obtained here for acid reduction and aldehyde oxidation by redox state (31). With this enzyme, the dithionite-reduced ES-1 AOR at 85ЊC. In fact, like ES-1 AOR, the acid reduction form exhibits a very complex signal which arises from the activity of C. thermoaceticum CAR was severely inhibited by spin-spin interactions of the S ϭ 3/2 center with a second low concentrations of reduced viologen (Յ170 ␮M) (52). Thus, paramagnetic center of lower reduction potential (31). Anal- the clostridial CARs and ES-1 AOR are also related by their ogous resonances but at much lower intensity can be seen in ability to catalyze a reversible reaction in vitro, and the same is the spectrum from ES-1 AOR near g ϭ 2.1 (Fig. 6). This presumably true for P. furiosus AOR and T. litoralis FOR, suggests that the second center is also present in the ES-1 although acid reduction by these enzymes has not been inves- enzyme but that it has a lower reduction potential than its tigated. counterpart in P. furiosus AOR, i.e., it is not reduced by so- As yet, a physiological function has not been unequivocally dium dithionite. The spectrum of ES-1 AOR shown in Fig. 6 defined for AOR or for the FOR and CAR enzymes. It has decreased in intensity with increasing temperature and was not been suggested (50) that CAR might function to oxidize acet- observed above 20 K, indicating a very rapid rate of spin aldehyde to acetate and provide low-potential reductant for relaxation. ES-1 AOR was EPR silent after oxidation of the CO2 reduction, but the source of acetaldehyde is not clear and dithionite-reduced form with excess thionin (E0Јϭϩ60 mV). the physiological electron carrier for CAR is unknown (46). CAR has also been proposed to reduce the carboxylic acids DISCUSSION generated as end products of carbohydrate fermentation in C. formicoaceticum (50), and it could also play a role in the uti- Thermococcus strain ES-1 AOR is the first aldehyde-oxidiz- lization of aromatic aldehydes (17). Concerning the P. furiosus ing enzyme to be isolated from an obligately proteolytic organ- AOR, it was speculated that it catalyzes the oxidation of glyc- ism. To date, three distinct types of aldehyde-oxidizing en- eraldehyde to glycerate in a novel Entner-Doudoroff pathway zymes, termed AOR (32), FOR (33, 37), and GAPOR (34), for sugar fermentation (32). Other enzyme reactions in the from the hyperthermophiles P. furiosus and T. litoralis have putative pathway were subsequently identified (41), but the been characterized. Both of these organisms, in contrast to existence of the pathway is not supported by recent nuclear ES-1, are able to use carbohydrates as well as proteinaceous magnetic resonance (20, 40) and enzymatic (34) analyses. Sim- materials as a carbon and energy source (14, 33, 35). The ilarly, the data presented here also argue against a glycolytic aldehyde-oxidizing enzyme of ES-1 is clearly distinct from role for AOR. ES-1 is not known to utilize sugars, and yet its

FOR and GAPOR. For example, FOR oxidizes only C1 to C3 AOR is virtually identical to the P. furiosus enzyme. Moreover, aliphatic aldehydes (33), while the only known substrate for glyceraldehyde was a very poor substrate for ES-1 AOR, as

GAPOR is GAP (34). On the other hand, ES-1 AOR closely shown by its low apparent kcat/Km value (Table 2). resembles P. furiosus AOR (10, 32) in its ability to oxidize both A clue to the physiological role of AOR in the hyperther- aliphatic and aromatic aldehydes; its electrophoretic and chro- mophiles may lie with the substrate speciﬁcity of the ES-1 matographic behavior; its contents of W, Fe, Mg, P, and cys- enzyme. The most effective substrates are acetaldehyde, iso- teine residues; and its susceptibility to inhibition by arsenite, valerylaldehyde, phenylacetaldehyde, and indoleacetaldehyde iodoacetate, and high concentrations of substrates. ES-1 and P. (apparent Km values of Ͻ100 ␮M), which are the aldehyde furiosus AORs also have similar EPR properties, which are derivatives of alanine, leucine, phenylalanine, and tryptophan, distinct from those of FOR (33). By analogy with P. furiosus respectively. These aldehydes also correspond to the substrates AOR, the predominant EPR signal of dithionite-reduced ES-1 for several ferredoxin-linked keto acid oxidoreductases that AOR (Fig. 6) can be assigned to a reduced S ϭ 3/2 [4Fe-4S]ϩ are found in ES-1 (27), T. litoralis, and P. furiosus (1, 2). These cluster (10), while the additional and much weaker EPR res- are pyruvate ferredoxin oxidoreductase (POR) (7), indolepyru- VOL. 177, 1995 TUNGSTEN-CONTAINING ALDEHYDE FERREDOXIN OXIDOREDUCTASE 4763

thesis, from S0, polysulfides, and aldehydes, of alkyl and aryl cyclic polysulfides, which are found at high concentrations in some Thermococcus spp. (39). However, the function of these compounds is not clear, and P. furiosus, which contains high AOR activity, grows well in the absence of S0. A role for AOR in acid reduction therefore appears unlikely. The great similarity in the catalytic properties of the AORs from proteolytic ES-1 and saccharolytic P. furiosus and the CARs of the mesophilic acetogens, suggests that all of these enzymes have the same function. For example, acetate is the major end product of carbohydrate fermentation by P. furiosus (14, 21), and it is generated from acetyl coenzyme A produced by POR. Similarly, the CAR-containing mesophile C. formicoaceticum also produces large amounts of acetate ultimately derived from pyruvate via POR (15). We therefore propose that AOR in the saccharolytic hyperthermophiles and CAR in the acetogens function to oxidize any acetaldehyde that is generated from pyruvate, either nonenzymatically or via the FIG. 7. Proposed role of AOR in the metabolism of ES-1. Modified from the POR reaction. In proteolytic organisms like ES-1, AOR also model of Ma et al. (27). Abbreviations: TA, transaminase; KAOR, 2-keto acid oxidizes additional aldehydes from the keto acids derived from ferredoxin oxidoreductase; ACS, acetyl coenzyme A synthetase; Fd, ferredoxin; amino acid oxidation. Further work is obviously required to CoASH, coenzyme A; Acyl CoA, acyl coenzyme A. substantiate this hypothesis, since with the data currently available, it is not possible to unambiguously define the physiological role of AOR. The scheme in Fig. 7 therefore encompasses vate ferredoxin oxidoreductase (IOR) (28, 29), and 2-ke- both a reductive function and an oxidative function for this toisovalerate ferredoxin oxidoreductase (VOR) (1, 2). POR, enzyme. A key aspect, for example, is to determine the extent VOR, and IOR preferentially utilize pyruvate, branched-chain to which aldehydes are by-products of the reaction catalyzed by 2-keto acids, and aromatic 2-keto acids, respectively. The acyl 2-keto acid oxidoreductases, both in vivo and in vitro. coenzyme A derivatives produced by these oxidoreductases can be utilized for ATP synthesis (42) to generate the corre- ACKNOWLEDGMENTS sponding acids (27). Hence, organic acids such as isovalerate, isobutyrate, and phenylacetate are excreted into the growth This research was supported by grants from the Department of medium by all of the proteolytic hyperthermophiles so far Energy (FG09-88ER13901), the Office of Naval Research (N00014- 90-J-1894), and the Deutsche Forschungsgemeinschaft. investigated (14, 35, 39, 44). The fact that the aldehyde substrates that are most readily oxidized by AOR are derivatives REFERENCES of amino acids strongly suggests that the former are generated 1. Adams, M. W. W. 1993. Enzymes and proteins from organisms that grow from the latter. In the absence of any obvious biosynthetic or near and above 100ЊC. Annu. Rev. Microbiol. 47:627–658. energetic benefit of such a reaction, aldehyde production is 2. Adams, M. W. W. 1994. Biochemical diversity among sulfur-dependent hy- assumed to be an intrinsic and obligate consequence of amino perthermophilic microorganisms. FEMS Microbiol. 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