| The goal of this research was to elucidate the catabolic pathway of peptide fermentation in hyperthermophilic archaea, a group of microorganisms able to grow optimally at 80{dollar}spcirc{dollar}C and above. In particular, the studies described here focused on identification and characterization of the enzymes catalyzing the formation of various organic acids, the end products of fermentation from their corresponding 2-keto acids, which are the transaminated products from various amino acids.; Three novel 2-keto acid ferredoxin oxidoreductases have been purified and characterized from the hyperthermophilic archaea, Pyrococcus furiosus, Thermococcus litoralis and Thermococcus strain ES-1. These are indolepyruvate ferredoxin oxidoreductase (IOR), 2-ketoisovalerate ferredoxin oxidoreductase (VOR) and 2-ketoglutarate ferredoxin oxidoreductase (KGOR), all of which are analogous to pyruvate ferredoxin oxidoreductase and catalyze the oxidative decarboxylation of 2-keto acids. IOR and VOR preferentially use as substrates the aromatic and branched-chain 2-keto acids, respectively, whereas KGOR is very specific for 2-ketoglutarate. IOR and VOR are the first examples of this type of oxidoreductase to be described while KGOR appears to be different from the one characterized previously from a mesophilic archaeon. Biochemical and spectroscopic characterizations of IOR, VOR and KGOR demonstrated that they are closely related and belong the same protein family. In addition, these are also iron-sulfur proteins and their catalytic mechanisms, which are distinct from that of pyruvate dehydrogenase, resemble that of a well-studied pyruvate ferredoxin oxidoreductase from a mesophilic archaeon.; Two unique ADP-dependent acetyl-coenzyme A (CoA) synthetase isoenzymes were also purified and characterized from hyperthermophilic archaeon, Pyrococcus furiosus. These differ in their substrate specificities and catalyze the formation of various organic acids from their corresponding CoA-derivatives, which are the products of the oxidoreductases described above. The molecular and catalytic properties of this type of enzyme are very different from the conventional AMP-forming acetyl-CoA synthetase found in many mesophilic microorganisms. |
