MOLECULAR AND PHYLOGENETIC CHARACTERIZATION OF PYRUVATE AND 2-KETOISOVALERATE FERREDOXIN OXIDOREDUCTASES FROM PYROCOCCUS-FURIOSUS AND PYRUVATE FERREDOXIN OXIDOREDUCTASE FROM THERMOTOGA-MARITIMA

Previous studies have shown that the hyperthermophilic archaeon Pyroco ccus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreduc tases (ORs) which differ in their substrate specificities, while the h yperthermophilic bacterium Thermotoga maritima contains only one, pyru vate ferredoxin oxidoreductase (FOR), These enzymes catalyze the synth esis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dep endent oxidative decarboxylation reaction with reduction of ferredoxin . We report here on the molecular analysis of the FOR (per) and 2-keto isovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the FOR gene from T. maritima, all of which comprise four differen t subunits, The operon organization for P. furiosus FOR and VOR was po rG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzym es, The operon organization for T. maritima POR was porGDAB, The three enzymes were 46 to 53% identical at the amino acid level, Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXC XXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amin o-terminal sequence comparisons show that FOR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogeneticall y homologous OR family, Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochon driate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequ ence identity). Three of these subunits are also homologous to the dim eric POR from a mesophilic archaeon, Halobacterium halobium (21% ident ity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.