title: 6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.
abstract: D-Ribulose-5-phosphate-3-epimerase (RPE) and 6-phosphofructokinase (PFK) catalyse two reactions in the ribulose monophosphate (RuMP) cycle in Bacillus methanolicus. The B. methanolicus wild-type strain MGA3 possesses two putative rpe and pfk genes encoded on plasmid pBM19 (rpe1-MGA3 and pfk1-MGA3) and on the chromosome (rpe2-MGA3 and pfk2-MGA3). The wild-type strain PB1 also encodes putative rpe and pfk genes on plasmid pBM20 (rpe1-PB1 and pfk1-PB1*); however, it only harbours a chromosomal pfk gene (pfk2-PB1). Transcription of the plasmid-encoded genes was 10-fold to 15-fold upregulated in cells growing on methanol compared to mannitol, while the chromosomal genes were transcribed at similar levels under both conditions in both strains. All seven gene products were recombinantly produced in Escherichia coli, purified and biochemically characterized. All three RPEs were active as hexamers, catalytically stimulated by Mg2+and Mn2+and displayed similar K' values (56-75 μM) for ribulose 5-phosphate. Rpe2-MGA3 showed displayed 2-fold lower Vmax(49 U/mg) and a significantly reduced thermostability compared to the two Rpe1 proteins. Pfk1-PB1* was shown to be non-functional. The PFKs were active both as octamers and as tetramers, were catalytically stimulated by Mg2+and Mn2+, and displayed similar thermostabilities. The PFKs have similar Kmvalues for fructose 6-phosphate (0.61-0.94 μM) and for ATP (0.38-0.82 μM), while Pfk1-MGA3 had a 2-fold lower Vmax(6.3 U/mg) compared to the two Pfk2 proteins. Our results demonstrate that MGA3 and PB1 exert alternative solutions to plasmid-dependent methylotrophy, including genetic organization, regulation, and biochemistry of RuMP cycle enzymes.
authors.affiliation: Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, Postboks 4760 Sluppen, 7465, Trondheim, Norway.
authors.forename: Simone Balzer
authors.initials: SB
authors.lastname: Le
authors.affiliation: Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, Postboks 4760 Sluppen, 7465, Trondheim, Norway.
authors.forename: Tonje Marita Bjerkan
authors.initials: TMB
authors.lastname: Heggeset
authors.affiliation: Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, Postboks 4760 Sluppen, 7465, Trondheim, Norway.
authors.forename: Tone
authors.initials: T
authors.lastname: Haugen
authors.affiliation: Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry, Postboks 4760 Sluppen, 7465, Trondheim, Norway.
authors.forename: Ingemar
authors.initials: I
authors.lastname: Nærdal
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Høgskoleringen 5, 7034, Trondheim, Norway. trygve.brautaset@ntnu.no.
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D000432:Methanol / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D012274:Ribulosephosphates / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012274:Ribulosephosphates; D008353:Mannitol; D010732:Phosphofructokinase-1; D002238:Carbohydrate Epimerases; C022553:ribulosephosphate 3-epimerase; D000432:Methanol
keywords: Characterization; Enzyme kinetics; Methylotrophy; Recombinant expression; RuMP cycle