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Comparative Study
.1999 Feb;181(4):1163-70.
doi: 10.1128/JB.181.4.1163-1170.1999.

An unusual oxygen-sensitive, iron- and zinc-containing alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

Affiliations
Comparative Study

An unusual oxygen-sensitive, iron- and zinc-containing alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

K Ma et al. J Bacteriol.1999 Feb.

Abstract

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degreesC by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 +/- 1,000, appears to be a homohexamer, and contains iron ( approximately 1.0 g-atom/subunit) and zinc ( approximately 1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23 degreesC. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95 degreesC of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9. 4 and 10.2 (at 80 degreesC), and the apparent Kms (at 80 degreesC) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.

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Figures

FIG. 1
FIG. 1
SDS-polyacrylamide electrophoresis gel (12.5%) of ADH purified fromP. furiosus. Lanes 1 and 4, molecular mass markers; lanes 2 and 3, 1 and 2 μg of ADH, respectively.
FIG. 2
FIG. 2
Amino-terminal amino acid sequences of ADHs from various sources. Abbreviations and references: Pf,P. furiosus (this work); ES,Thermococcus strain ES-1 (37); Tl,T. litoralis (34); Tz,T. zilligii (31); Ss,Sulfolobus solfataricus (2); Zm,Z. mobilis (adh2 [46]); Tb,Thermoanaerobium brockii (47); Sc,Saccharomyces cerevisiae (5); Dm,Drosophila melanogaster (68); Ml,Methanogenium liminatans (8). Residues identical to those in theP. furiosus enzyme are in boldface. X, unidentified residue.
FIG. 3
FIG. 3
EPR spectra ofP. furiosus ADH. ADH was used at a concentration of 3.6 mg/ml. a, ADH as isolated in 50 mM Tris-HCl (pH 7.8) containing 5% (vol/vol) glycerol and 2 mM dithiothreitol. Spectrum was recorded at 4 K with 5 mW of microwave power. b, ADH treated with NO. The enzyme (3.6 mg/ml in 50 mM Tris-HCl [pH 7.8]) was gently bubbled with NO for 3 min at 25°C prior to being frozen in liquid N2. The spectrum was recorded with 40 mW of microwave power at 4 K. The spectrometer settings were as follows: microwave frequency, 9.597 GHz; modulation frequency, 100 kHz; modulation amplitude, 5 G; time constant, 163.84 ms; gain, 2 × 105; and scale, 16.
FIG. 4
FIG. 4
Effects of temperature on the stability ofP. furiosus ADH. The enzyme (3.6 mg/ml in 50 mM Tris-HCl [pH 7.8] containing 2.0 mM dithiothreitol) was incubated in stoppered glass vials at 85°C (●) or 95°C (□). Samples were removed at intervals and assayed by the NADP-dependent oxidation of ethanol at 80°C.
FIG. 5
FIG. 5
Proposed metabolic role ofP. furiosus ADH. Abbreviations: Fdox, oxidized ferredoxin; Fdred, reduced ferredoxin; KAOR, α-keto acid ferredoxin oxidoreductases; CoASH, coenzyme A; AOR, aldehyde ferredoxin oxidoreductase; ACS, acetyl-CoA synthetases; FNOR, ferredoxin NADP oxidoreductase; SH, sulfhydrogenase (or hydrogenase).
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