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

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Abstract
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.