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Methods in enzymology

Characterization of the recombinant pyrophosphate-dependent 6-phosphofructokinases from Methylomicrobium alcaliphilum 20Z and Methylococcus capsulatus Bath.


PMID 21419911

Abstract

The Embden-Meyerhof-Parnas (EMP) glycolysis is the starting point of the core carbon metabolism. Aerobic methanotrophs possessing activity of the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the classical glycolytic enzyme ATP-dependent 6-phosphofructokinase (ATP-PFK) are promising model bacteria for elucidation of the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in microorganisms. Characterization of the His(6)-tagged PPi-PFKs from two methanotrophs, halotolerant alkaliphilic Methylomicrobium alcaliphilum 20Z and thermotolerant Methylococcus capsulatus Bath, showed differential capabilities of PPi-PFKs to phosphorylate sedoheptulose-7-phosphate and this property correlated well with the metabolic patterns of these bacteria assimilating C(1) substrate either via the ribulosemonophosphate (RuMP) pathway (Mm. alcaliphilum 20Z) or simultaneously via the RuMP and serine pathways and the Calvin cycle (Mc. capsulatus Bath). Analysis of the genomic draft of Mm. alcaliphilum 20Z (https://www.genoscope.cns.fr/agc/mage) has provided in silico evidence for the existence of a PPi-dependent pyruvate-phosphate dikinase (PPDK). Expression of the ppdk gene at oxygen limitation along with the presence of PPi-PFK in Mm. alcaliphilum 20Z implied functioning of PPi-dependent glycolysis and PPi recycling under conditions when oxidative phosphorylation is hampered.