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

Abstract

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.

Keywords: Citrus sinensis; Fructose-2,6-bisphosphate; Hetero-hexamer; Orange; PPi-dependent phosphofructokinase; Recombinant protein.