We report the first enzymatic synthesis of <smlcap>D</smlcap>-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 31P and 1H 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 <smlcap>D</smlcap>-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TFHis6) of Escherichia coli. The active fusion enzyme was named TagK-TFHis6. Tag-1P and <smlcap>D</smlcap>-fructose-1-phosphate are substrates for the TagK-TFHis6 enzyme, whereas the isomeric derivatives <smlcap>D</smlcap>-tagatose-6-phosphate and <smlcap>D</smlcap>-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.

Journal Section:
Research Article
Keywords:
Tagatose, Phosphoenolpyruvate:sugar phosphotransferase system, Tagatose-1-phosphate kinase, Bacillus licheniformis, Klebsiella pneumoniae, 31P and 1H nuclear magnetic resonance, Nuclear magnetic resonance characterization of tagatose phosphate
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