Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases fromThermotoga maritimaandThermotoga hypogea

Abstract

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.

A novel bifunctional aldehyde/alcohol dehydrogenase mediating ethanol formation from acetyl-CoA in hyperthermophiles

“…At present, research has been focused on the identification of hyperthermophilic enzymes involved in the ethanol fermentation pathway. The pyruvate ferredoxin oxidoreductases (POR or PFOR) from T. maritima (Tma) and T. hypogea (Thy) were found to have CoAdependent pyruvate decarboxylase (PDC) activity, where the PDC activity is less than 2% of POR activity [19,20]. Thus, more than 98% of pyruvate is probably converted to ac-CoA by POR, and ac-CoA is further converted to acetic acid if there is no enzyme to catalyze the reduction of ac-CoA to produce ac-ald (Fig.…”

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

“…PDC activity was assayed by measuring the rate of the production of acetaldehyde from pyruvate [7] , [8] . The final concentration of acetaldehyde and isobutyraldehyde were determined using a calibration curve prepared by linear regression plotting of known concentrations of each product processed under the same assay conditions.…”

“…One such enzyme was discovered to be the bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreductase (PDC/POR) and was described the first time in the hyperthermophilic archaeon Pyrococcus furiosus [6] . The bifunctional enzyme is shown to be able to catalyze both non-oxidative and oxidative decarboxylation of pyruvate to produce acetaldehyde and acetyl-CoA, respectively [6] , [7] , [8] and the bifunctionality seems to be a universal trait of hyperthermophilic (archaeal and bacterial) PORs.…”

Optimization of expression and properties of the recombinant acetohydroxyacid synthase of Thermotoga maritima

“…Standard procedures were followed for all DNA manipulation, competent cell preparation and transformation according to the methods described by Sambrook and Russell [2] . DNA was isolated from Thermotoga maritima biomass that was grown anaerobically on glucose and yeast extract at 80 °C as described by Huber et al [3] with modifications as previously described [4] . The medium contained (per liter) KCl, 2 g; MgCl 2 ·6H 2 O, 1.42 g; MgSO 4 ·7H 2 O, 1.8 g; CaCl 2 ·2H 2 O, 0.05 g; NaCl, 20 g; (NH4) 2 CO 3 , 1.14 g; KH 2 PO 4 , 0.05 g; resazurin 0.05 mg, trace minerals as described previously by Balch et al [5] , 10 ml; yeast extract, 2.5 g; and glucose, 4.0 g. Before autoclave the pH of the medium was adjusted to 6.8 using 1 M NaOH.…”