Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg−1 and 20.2 ± 1.8 U mg−1, with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic β-keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding β-keto acids.

The bifunctional alcohol/ aldehyde dehydrogenase enzyme in Entamoeba histolytica (EhADH2) belongs to the ADHE iron dependent family, and is essential for trophozoite growth and survival. EhADH2 catalyzes the conversion of acetyl Co‐A to acetaldehyde and the final reduction of acetaldehyde to ethanol by its separate ADH and ALDH domains respectively. The primary focus of research was to determine the effect of alternative treatments for amebiasis through inhibition by pyrazoline inhibitors on the EhADH2 enzyme. Series 1a (1,3‐diphenyl‐1‐carbamoyl‐2‐pyrazoline), series 1b (3‐diphenyl‐1‐propylcarbamoyl‐2‐pyrazoline), series 2 (1,3,4‐triphenyl‐1‐carbamoyl‐2‐pyrazoline), pyrazoline derivatives showed capabilities to inhibit EhADH2 activities. A kinetic assay was performed to calculate the Ki value for inhibitor [3‐(4‐chlorophenyl)‐1‐(bromophenylcarboxamide)‐2‐pyrazoline], inhibitor [3‐4(‐chlorophenyl)‐1‐N‐propylcarboxamide‐2‐pyrazoline], and inhibitor [3‐(4‐cholorphenyl)‐4‐phenyl‐1‐(4‐chlorophenyl carboxamide)‐2‐pyrazoline] at the rate of enzymatic affinity with the substrate acetaldehyde at NAD+ μM/min as the unit of measurement; then, the Ki value for the inhibitor was calculated using Michaelis‐Menten and Lineweaver‐Burke equations. Inhibitor [3‐(4‐chlorophenyl)‐4‐phenyl‐1‐(4‐chlorophenyl carboxamide)‐2‐pyrazoline], from series 2, demonstrated higher inhibition of EhADH2 enzymatic activities compared to series 1a and 1b. Future modifications to the basic structures of the three series shall improve the efficiency of these compounds.

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