Abstract
2-ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of aliphatic amino acid-derived 2-keto acids. The enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (αβγδ)2 consisting of four different subunits, α = 45 kDa, β = 31 kDa, γ = 22 kDa and δ = 13 kDa, respectively, and it has three [4Fe-4S] clusters per αβγδ-protomer, similar to other ferredoxin oxidoreductases. In the present study, the native enzyme was purified from this strain and crystallized to give rod-like crystals that were suitable for X-ray diffraction experiments. The crystals belonged to space group P41212, with unit-cell parameters a = b = 136.20 A, c = 221.07 A. Diffraction images were processed to a resolution of 3.0 A. The data collected so far indicate the approximate molecular boundaries and a partial main-chain trace of the enzyme.
Highlights
An energy-producing pathway in the hyperthermophilic archaeon, Thermococcus profundus, has been proposed to degrade amino acids [1] [2]
Amino acids produced from some peptides by peptidases are converted to their corresponding 2-keto acids by transaminases
Pyruvate ferredoxin oxidoreductase (PFOR) is one of the well-characterized members of the ferredoxin-dependent enzyme family [3]-[5], and it has been shown to catalyze the production of acetaldehyde in the presence of coenzyme A [6]
Summary
An energy-producing pathway in the hyperthermophilic archaeon, Thermococcus profundus, has been proposed to degrade amino acids [1] [2]. In this pathway, amino acids produced from some peptides by peptidases are converted to their corresponding 2-keto acids by transaminases. The 2-ketoisovalerate ferredoxin oxidoreductase (VOR) catalyzes a reaction to produce acetyl CoA in the presence of coenzyme A from these 2-keto acids through oxidative decarboxylation. In order to investigate the structure-function relationships and deduce specific substrate recognition mechanisms of these four types of enzyme (PFOR, VOR, KGOR, IOR), we report here the purification and crystallization of VOR from Thermococcus profundus and a preliminary analysis of crystallographic data collected for this enzyme
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