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

Abstract

Pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) was obtained as His 6-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 C 1 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.