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

Background: The phosphoenolpyruvate (PEP): lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICBLac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. Results: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-beta-D-galactopyranoside and EIICBLac with UV light caused a loss of EIICBLac activity. Nevertheless, photoin-activated EIICBLac could still be phosphorylated with [P-32]PEP. Proteolysis of photoinactivated [P-32]P-EIICBLac 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 [P-32]P similar to EIICBLac, photoinactivated [P-32]P-EIICBLac remained strongly phosphorylated. Conclusion: Phosphorylation of the -OH group at C6 of p-nitrenephenyl-beta-D-galactopyranoside covalently bound to EIICLac by the histidyl-phosphorylated [P-32]P similar to EIIBLac domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-beta-D-galactopyranoside into the active site of modelled EIICLac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained. (c) 2018 S. Karger AG, Basel