title: Characterization of recombinant pyrophosphate-dependent 6-phosphofructokinase from halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.
abstract: 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.
authors.affiliation: Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Prospect Nauki 5, Pushchino 142290, Moscow, Russia.
authors.forename: Olga N
authors.initials: ON
authors.lastname: Rozova
authors.forename: Valentina N
authors.initials: VN
authors.lastname: Khmelenina
authors.forename: Stéphane
authors.initials: S
authors.lastname: Vuilleumier
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Trotsenko
mesh_terms: D016000:Cluster Analysis; D011756:Diphosphates / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D008772:Methylococcaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism
chemical_list: D011756:Diphosphates; D020536:Enzyme Activators; D005635:Fructosediphosphates; D005636:Fructosephosphates; D011993:Recombinant Fusion Proteins; D013403:Sugar Phosphates; C020495:sedoheptulose 7-phosphate; C027618:fructose-6-phosphate; D010732:Phosphofructokinase-1; C029063:fructose-1,6-diphosphate