BackgroundThe efficient production of sustainable biofuels is important for the reduction of greenhouse gas emissions.Clostridium thermocellumATCC 27405 is a candidate for ethanol production from lignocellulosic biomass using consolidated bioprocessing. Fermentation of cellulosic biomass goes through an atypical glycolytic pathway in this thermophilic bacterium, with various glycolytic enzymes capable of utilizing different phosphate donors, including GTP and inorganic pyrophosphate (PPi), in addition to or in place of the usual ATP.C.thermocellumcontains three annotated phosphofructokinases (PFK) genes, the expression of which have all been detected through proteomics and transcriptomics. Pfp (Cthe_0347) was previously characterized as pyrophosphate dependent with fructose-6-phosphate (F6P) as its substrate.ResultsWe now demonstrate that this enzyme can also phosphorylate sedoheptulose-7-phosphate (an intermediate in the pentose phosphate pathway), with theVmaxandKmof F6P being approximately 15 folds higher and 43 folds lower, respectively, in comparison to sedoheptulose-7-phosphate. Purified PfkA shows preference for GTP as the phosphate donor as opposed to ATP with a 12.5-fold difference inKmvalues while phosphorylating F6P. Allosteric regulation is a factor at play in PfkA activity, with F6P exhibiting positive cooperativity, and an apparent requirement for ammonium ions to attain maximal activity. Phosphoenolpyruvate and PPiwere the only inhibitors for PfkA determined from the study, which corroborates what is known about enzymes from this subfamily. The activation or inhibition by these ligands lends support to the argument that glycolysis is regulated by metabolites such as PPiand NH4+in the organism. PfkB, showed no activity with F6P, but had significant activity with fructose, while utilizing either ATP or GTP, making it a fructokinase. Rounding out the upper glycolysis pathway, the identity of the fructose-1,6-bisphosphate aldolase in the genome was verified and reported to have substantial activity with fructose-1,6-bisphosphate, in the presence of the divalent ion, Zn2+.ConclusionThese findings along with previous proteomic data suggest that Pfp, plays a role in both glycolysis and the pentose phosphate pathway, while PfkA and PfkB may phosphorylate sugars in glycolysis but is responsible for sugar metabolism elsewhere under conditions outside of growth on sufficient cellobiose.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02362-8.