pyrophosphate and sedoheptulose-7-phosphate

Pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) was obtained as His₆-tagged protein by cloning of the pfp gene from the aerobic obligate methanotroph Methylomicrobium alcaliphilum 20Z and characterized. The recombinant PPi-PFK (4×45 kDa) was highly active, non-allosteric and stringently specific to pyrophosphate as the phosphoryl donor. The enzyme was more specific for the reverse reaction substrate fructose-1,6-bisphosphate (K(m) 0.095 mM, V(max) 805 U/mg of protein) than for the forward reaction substrate fructose-6-phosphate (K(m) 0.64 mM, V(max) 577 U/mg of protein). It also phosphorylated sedoheptulose-7-phosphate with much lower efficiency (K(m) 1.01 mM, V(max) 0.118 U/mg of protein). The kinetic properties of the M. alcaliphilum PP(i)-PFK were analyzed and compared with those of PP(i)-PFKs from other methanotrophs. The PP(i)-PFK from M. alcaliphilum shows highest sequence identity to PPi-PFK from obligate mesophilic methanotroph Methylomonas methanica (89%), and only low identity to the enzyme from thermotolerant Methylococcus capsulatus Bath (16%). This extensive sequence divergence of PPi-PFKs correlated with differential ability to phosphorylate sedoheptulose-7-phosphate and with the metabolic patterns of these bacteria assimilating C₁ substrate either via the ribulose monophoshate (RuMP) cycle or simultaneously via the RuMP and the Calvin cycles. Based on enzymic and genomic data, the involvement of PPi-PFK in pyrophosphate-dependent glycolysis in M. alcaliphilum 20Z was fist proposed.

Topics: Cluster Analysis; Diphosphates; Enzyme Activators; Fructosediphosphates; Fructosephosphates; Kinetics; Methylococcaceae; Molecular Weight; Phosphofructokinase-1; Phylogeny; Protein Multimerization; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Sugar Phosphates