Purification and properties of the pyrophosphate-dependent phosphofructokinase from Dictyoglomus thermophilum Rt46 B.1

The distribution of phosphofructokinase phosphoryl donor subtypes (ATP-, AD P-, and pyrophosphate) in the deeply rooted phylogenetic lineages of thermo philes is of interest with regard to the evolution of phosphofructokinase a ctivity and of the Embden-Meyerhof pathway. In this article we present the first biochemical description of a thermostable pyrophosphate-dependent pho sphofructokinase from the hyperthermophilic bacterium Dictyoglomus thermoph ilum. The enzyme was not allosterically controlled by traditional modulator s of phosphofructokinases and has significant activity with tripolyphosphat e and polyphosphate. Kinetic parameters of the enzyme suggest it plays prim arily a glycolytic role. The enzyme required Mg2+ for optimal activity, was partially activated by some monovalent and divalent cations, and was stron gly inhibited by CU2+. The sequence of the 21 N-terminal residues suggests that the enzyme is most similar to the pyrophosphate-dependent phosphofruct okinases from Amycolatopsis methanolica and the hyperthermophilic crenarcha eon Thermoproteus tenax, enzymes which have been suggested to represent an ancient lineage of phosphofructokinases (Siebers et al. 1998). The unexpect ed finding of a pyrophosphate-dependent phosphofructokinase in Dictyoglomus thermophilum, which is phylogenetically related to Thermotoga maritima, pr eviously shown to possess an ATP-dependent phosphofructokinase activity, is discussed.