title: Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus.
abstract: The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX(C)) and one on plasmid pBM19 (GlpX(P)), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpX(C) and glpX(P) from B. methanolicus. GlpX(P) and GlpX(C) share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn(2+) ions and inhibited by Li(+), but differed in terms of the kinetic parameters. GlpX(C) showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s(-1) mM(-1) and 14 ± 0.5 μM, respectively) than GlpX(P) (8.8 s(-1) mM(-1) and 440 ± 7.6 μM, respectively), indicating that GlpX(C) is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX(P) but not for GlpX(C). Based on these in vitro data, GlpX(P) is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.
authors.affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology.
authors.forename: Jessica
authors.initials: J
authors.lastname: Stolzenberger
authors.forename: Steffen N
authors.initials: SN
authors.lastname: Lindner
authors.forename: Marcus
authors.initials: M
authors.lastname: Persicke
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
authors.forename: Volker F
authors.initials: VF
authors.lastname: Wendisch
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002876:Chromosomes, Bacterial; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D007700:Kinetics; D008094:Lithium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D008345:Manganese; D008094:Lithium; D006597:Fructose-Bisphosphatase; D010744:Phosphoric Monoester Hydrolases; C022627:sedoheptulose-bisphosphatase