6-Phosphofructokinase and ribulose-5-phosphate 3-epimerase in methylotrophic Bacillus methanolicus ribulose monophosphate cycle.
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 V max (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 K m values 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 V max (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.
['Le|Simone Balzer|SB|', 'Heggeset|Tonje Marita Bjerkan|TMB|', 'Haugen|Tone|T|', 'Nærdal|Ingemar|I|', 'Brautaset|Trygve|T|']
D001407:Bacillus D001426:Bacterial Proteins D002238:Carbohydrate Epimerases D002876:Chromosomes, Bacterial D004926:Escherichia coli D007700:Kinetics D008353:Mannitol D053858:Metabolic Networks and Pathways D000432:Methanol D010732:Phosphofructokinase-1 D010957:Plasmids D011994:Recombinant Proteins D012274:Ribulosephosphates D017422:Sequence Analysis, DNA
Characterization Methylotrophy RuMP cycle Enzyme kinetics Recombinant expression