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

• Published in: Journal of molecular microbiology and biotechnology Vol. 25; no. 2-3; p. 106
• Main Authors: Van der Heiden, Edwige, Delmarcelle, Michaël, Simon, Patricia, Counson, Melody, Galleni, Moreno, Freedberg, Darón I, Thompson, John, Joris, Bernard, Battistel, Marcos D
• Format: Journal Article
• Language: English
• Published: Switzerland 01-01-2015
• Subjects: Bacillus - enzymology; Bacillus - metabolism; Escherichia coli - genetics; Fructose - metabolism; Fructosephosphates - metabolism; Hexosephosphates - metabolism; Hexoses - chemistry; Hexoses - metabolism; Klebsiella pneumoniae - enzymology; Klebsiella pneumoniae - growth & development; Magnetic Resonance Spectroscopy; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors; Phosphotransferases (Alcohol Group Acceptor) - isolation & purification; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Substrate Specificity
• ISSN: 1660-2412
• DOI: 10.1159/000370115

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.