An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C 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 (∼1.0 g-atom/subunit) and zinc (∼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°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent Km s (at 80°C) 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.

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii

“…The central metabolism of this organism uses an unusual NADPH-dependent sulfide dehydrogenase which is capable of both the NADPH-dependent reduction of elemental sulfur and the NADP + -dependent oxidation of ferredoxin [22]. A unique NADPH-dependent alcohol dehydrogenase with wide substrate specificity and a strong preference for the reduction of aldehydes to alcohols is also found in this organism [23]. This unusual mix of NADH and NADPH dependent reactions in catabolic processes may account for the finding that the pyrococcal CoADR, which would be expected to show a strong preference for NADPH, is able to efficiently utilize either reduced nucleotide substrate.…”

The Fur iron regulator-like protein is cryptic in the hyperthermophilic archaeonThermococcus kodakaraensis

“…It is a cofactor for many enzymes and is involved in vital metabolic processes. Archaeal genomes code for a high number of putative proteins predicted to contain iron, including many hydrogenases and Fe-S cluster proteins (Ma & Adams, 1999;Dobbek et al, 2001;Kanai et al, 2003;Major et al, 2004;Ferrer et al, 2007), which suggest the important role that this element plays. However, the need for iron of these organisms remains undefined, especially given their respective extreme natural habitats.…”

A Hyperactive NAD(P)H:Rubredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…This exhibited peaks near 375 and 450 nm which decreased in intensity upon the addition of sodium dithionite or NADPH (data not shown). As determined by using a molar absorptivity of 11,300 M Ϫ1 cm Ϫ1 at 450 nm (2), the holoenzyme contains one flavin moiety per mole, while plasma emission spectroscopy (13) showed the presence of 1.8 g-atoms of P per mol of enzyme. These data suggest that NROR contains one flavin adenine dinucleotide (FAD) molecule per mole.…”