title: Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.
abstract: We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.
authors.affiliation: Center for Protein Engineering, University of Liège, Institut de Chimie, Sart-Tilman, Belgium.
authors.forename: Edwige
authors.initials: E
authors.lastname: Van der Heiden
authors.forename: Michaël
authors.initials: M
authors.lastname: Delmarcelle
authors.forename: Patricia
authors.initials: P
authors.lastname: Simon
authors.forename: Melody
authors.initials: M
authors.lastname: Counson
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.forename: Darón I
authors.initials: DI
authors.lastname: Freedberg
authors.forename: John
authors.initials: J
authors.lastname: Thompson
authors.forename: Bernard
authors.initials: B
authors.lastname: Joris
authors.forename: Marcos D
authors.initials: MD
authors.lastname: Battistel
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005632:Fructose / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D006600:Hexosephosphates / Q000378:metabolism; D006601:Hexoses / Q000737:chemistry* / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000254:growth & development; D009682:Magnetic Resonance Spectroscopy; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005636:Fructosephosphates; D006600:Hexosephosphates; D006601:Hexoses; C032284:fructose-1-phosphate; D005632:Fructose; C026634:tagatose 6-phosphate; C027618:fructose-6-phosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017853:Phosphotransferases (Alcohol Group Acceptor); C032258:tagatose kinase; C030192:tagatose