Heterologous expression and kinetic characterization of the α, β and αβ blend of the PPi-dependent phosphofructokinase from Citrus sinensis

•Heterologous expression of a plant pyrophosphate-dependent phosphofructokinase.•Kinetic characterization of orange PPi-PFK and its regulation by Fru2,6bisP.•Fru2,6bisP activates the hetero-hexamer while α is not affected and β is inhibited.•The α3β3 hexamer is three orders of magnitude more active than each single subunits. This work reports the molecular cloning and heterologous expression of the genes coding for α and β subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg−1 for α and β subunits, respectively). On the other hand, co-expression rendered a α3β3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.