Characterization of recombinant pyrophosphate-dependent 6-phosphofructokinase from halotolerant methanotroph Methylomicrobium alcaliphilum 20Z

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 (Km 0.095 mM, Vmax 805 U/mg of protein) than for the forward reaction substrate fructose-6-phosphate (Km 0.64 mM, Vmax 577 U/mg of protein). It also phosphorylated sedoheptulose-7-phosphate with much lower efficiency (Km 1.01 mM, Vmax 0.118 U/mg of protein). The kinetic properties of the M. alcaliphilum PPi-PFK were analyzed and compared with those of PPi-PFKs from other methanotrophs. The PPi-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.