title: Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Methylococcus capsulatus Bath.
abstract: An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K(m) 2.27 mM and V(max) 7.6 U mg(-1) of protein), sedoheptulose-7-phosphate (K(m) 0.027 mM and V(max) 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K(m) for PPi was 0.027 mM, while in the reverse reaction, K(m) for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V(max) 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H(+)-translocating pyrophosphatase (H(+)-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.
authors.affiliation: G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, Moscow region, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Reshetnikov
authors.forename: Olga N
authors.initials: ON
authors.lastname: Rozova
authors.forename: Valentina N
authors.initials: VN
authors.lastname: Khmelenina
authors.forename: Ildar I
authors.initials: II
authors.lastname: Mustakhimov
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Beschastny
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Trotsenko
mesh_terms: D003001:Cloning, Molecular; D043564:Inorganic Pyrophosphatase / Q000235:genetics; D007700:Kinetics; D020619:Methylococcus capsulatus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D009876:Operon; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D011994:Recombinant Proteins; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D043564:Inorganic Pyrophosphatase