Photoinactivation of the Staphylococcus aureus Lactose-Specific EIICB Phosphotransferase Component with p-azidophenyl-β-D-Galactoside and Phosphorylation of the Covalently Bound Substrate

Abstract

<b><i>Background:</i></b> The phosphoenolpyruvate (PEP):lactose phosphotransferase system of<i> Staphylococcus aureus</i> transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICB^Lac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. <b><i>Results:</i></b> Irradiation of a reaction mixture containing the photoactivatable <i>p</i>-azidophenyl-β-D-galactopyranoside and EIICB^Lac with UV light caused a loss of EIICB^Lac activity. Nevertheless, photoinactivated EIICB^Lac could still be phosphorylated with [³²P]PEP. Proteolysis of photoinactivated [³²P]P-EIICB^Lac with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245–247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [³²P]P∼EIICB^Lac, photoinactivated [³²P]P-EIICB^Lac remained strongly phosphorylated. <b><i>Conclusion:</i></b> Phosphorylation of the –OH group at C6 of <i>p</i>-nitrenephenyl-β-D-galactopyranoside covalently bound to EIIC^Lac by the histidyl-phosphorylated [³²P]P∼EIIB^Lac domain is a likely explanation for the observed acid resistance. Placing <i>p</i>-nitrenephenyl-β-D-galactopyranoside into the active site of modelled EIIC^Lac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.