PP(i)-Dependent Phosphofructokinase from Thermoproteus tenax, an Archaeal Descendant of an Ancient Line in Phosphofructokinase Evolution

Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PP(i)-dependent PFK (PP(i)-PFK; EC 2.7.1.90), which uses PP(i) instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PP(i)-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PP(i)-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PP(i)-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PP(i)-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PP(i) as a cosubstrate.