Synthesis and Physicochemical Characterization of D-Tagatose-1-phosphate: The Substrate of the Tagatose-1-Phosphate Kinase TagK in the PTS-mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis

摘要

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multi-component PEP-dependent:tag-PTS present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by ³¹P and ¹H NMR 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 (Fru-1P) are substrates for the TagK-TF^His6 enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate (Tag-6P) and D-fructose-6-phosphate (Fru-6P) are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific enzyme II (EII^Tag) in E.coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and EI, to restore the phosphate transfer is demonstrated.