Abstract
Pyrococcus furiosus is a strictly anaerobic archaeon that grows optimally at 100 degrees C by a fermentative-type metabolism in which complex peptide mixtures such as yeast extract and Tryptone, and also certain sugars, are oxidized to organic acids, H2 and CO2. Enzymes involved in the utilization of peptides such as proteases, aromatic amino transferases, and glutamate dehydrogenase have been previously purified from this organism. It is shown here that P. furiosus also contains significant cytoplasmic concentrations of a new enzyme termed indolepyruvate ferredoxin oxidoreductase (IOR). This catalyzes the oxidative decarboxylation of aryl pyruvates, which are generated by the transamination of aromatic amino acids, to the corresponding aryl acetyl-CoA. IOR is a tetramer (alpha 2 beta 2) of two identical subunits (66,000 and 23,000 Da) with a molecular weight of 180,000. The enzyme contains one molecule of thiamine pyrophosphate and four [4Fe-4S]2+,1+ and one [3Fe-4S]0,1+ cluster, as determined by iron analyses and EPR spectroscopy. Significant amounts of other metals such as copper and zinc were not detected. IOR was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.5-10.5 (at 80 degrees C). The enzyme was sensitive to inactivation by O2, losing 50% of its activity after exposure to air for 20 min at 23 degrees C, and was quite thermostable, with a half-life of activity at 80 degrees C (under anaerobic conditions) of about 80 min. The Km values (in microM) for indolepyruvate, p-hydroxyphenylpyruvate, phenylpyruvate, CoASH, and P. furiosus ferredoxin, the physiological electron carrier, were 250, 110, 90, 17, and 48, respectively. IOR was inhibited by KCN (apparent Ki = 7.5 mM), but not by CO (1 atm). An enzyme analogous to IOR has not been reported previously. Curiously, it has few properties in common with the pyruvate ferredoxin oxidoreductase of P. furiosus, even though the two enzymes catalyze virtually identical reactions. In fact, of known ketoacid oxidoreductases, the catalytic mechanism of IOR appears to be most similar to that of the pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium Thermotoga maritima.
Highlights
HYPERTHERMOPHILIC ARCHAEAThese organisms convert the aryl 2-keto concentrations were routinely estimated by the Lowry method using acids generated by transamination reactions to thecorresponding aryl acetates eitherby direct oxidative decarboxylation or by decarboxylation t o the corresponding aldehyde followed by its oxidation
From the Department of Biochemistry and the Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602
It is shown here that E? furiosus contains significant cytoplasmic concentrations of a new enzymetermed indolepyruvate ferredoxin oxidoreductase (IOR).This catalyzesthe oxidative decarboxylation of aryl pyruvates, which are generated by thetransamination of aromatic amino acids, to the corresponding aryl acetyl-coA
Summary
These organisms convert the aryl 2-keto concentrations were routinely estimated by the Lowry method using acids generated by transamination reactions to thecorresponding aryl acetates eitherby direct oxidative decarboxylation or by decarboxylation t o the corresponding aldehyde followed by its oxidation. Thebuffer al., 1970),and indolepyruvate decarboxylase was recently char- used throughout the purification was mM Tris/HCI, pH 8.0, containacterized from Enterobacter cloacae Furiosus, of a new enzyme ining glycerol (lo%, v/v), sodiumdithionite (2 mM), anddithiothreitol (DTT 2mM).All chromatography columns were run using a fast protein liquidchromatographysystem(Pharmacia Biotech Inc.).The procedures for lysing the cells and preparing the cell-free extract were the same as for the purification of I? Furiosus differs from mesophilic bacteria in directly generating the acactivity elutedat 170-198 mM NaCl using a gradient (9 litefros)m 0 to 500 mM NaCl in buffer. The concentrated sampleof IOR from the previous step was applied to a column (6x 60 cm)of Superdex 200 (Pharmacia) equilibrated a5t
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