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 <smlcap>D</smlcap>-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of <i>Klebsiella pneumoniae</i>. 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 <i>Bacillus licheniformis</i> PTS-mediated <smlcap>D</smlcap>-tagatose catabolic pathway (<i>Bli</i>-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF^His6) of <i>Escherichia coli</i>. The active fusion enzyme was named TagK-TF^His6. Tag-1P and <smlcap>D</smlcap>-fructose-1-phosphate are substrates for the TagK-TF^His6 enzyme, whereas the isomeric derivatives <smlcap>D</smlcap>-tagatose-6-phosphate and <smlcap>D</smlcap>-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (k_cat/K_m) 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 <i>B. licheniformis</i> tagatose-specific Enzyme II in <i>E. coli</i> is inefficient. The capability of the PTS general cytoplasmic components of <i>B. subtilis</i>, HPr and Enzyme I to restore the phosphate transfer is demonstrated.