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J Mol Microbiol Biotechnol ; 25(2-3): 106-19, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26159072

RESUMO

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.


Assuntos
Bacillus/metabolismo , Hexoses/química , Hexoses/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Bacillus/enzimologia , Escherichia coli/genética , Frutose/metabolismo , Frutosefosfatos/metabolismo , Hexosefosfatos/metabolismo , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/crescimento & desenvolvimento , Espectroscopia de Ressonância Magnética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Especificidade por Substrato
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