Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles.

An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l -threonine dehydrogenases with binding motifs of catalytic zinc and NADP ⁺ . Metal analyses revealed that this NADP ⁺ -dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic ( R / S )-acetoin to (2 R ,3 R )-2,3-butanediol and meso -2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent K _m values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP ⁺ , respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation.

An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80°C. The V _max was 230 ± 14 μmol/min/mg ( k _cat / K _m = 548,000 min ⁻¹ mM ⁻¹ ), and the K _m values for NADH and oxygen were 42 ± 3 and 43 ± 4 μM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima .

A thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract ofThermotoga maritima. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purifiedT. maritimathioredoxin reductase could not use thioredoxin fromSpirulinaas an electron acceptor, it used thioredoxin that was purified fromT. maritimaby monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparentV_maxvalues of 1,111 ± 35 μmol NADH oxidized min⁻¹mg⁻¹and 115 ± 2.4 μmol NADPH oxidized min⁻¹mg⁻¹, respectively. The apparentK_mvalues were determined to be 89 ± 1.1 μM, 73 ± 1.6 μM, and 780 ± 20 μM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95°C, and more than 60% of the activity remained after incubation for 28 h at 80°C. The purifiedT. maritimathioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities.T. maritimathioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5′-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.