Supplementary Material for: 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 <b><i>Bacillus licheniformis</i></b>
2015-07-09T00:00:00Z (GMT) by
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 <i>Klebsiella pneumoniae</i>. Physicochemical characterization by <sup>31</sup>P and <sup>1</sup>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 <i>Bacillus licheniformis</i> PTS-mediated D-tagatose catabolic pathway (<i>Bli</i>-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF<sup>His6</sup>) of <i>Escherichia coli</i>. The active fusion enzyme was named TagK-TF<sup>His6</sup>. Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF<sup>His6</sup> enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub>) 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.