6-Phospho-β-galactosidases of Gram-positive and 6-phospho-β-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus
The 6-phospho-β-galactosidase of Staphylococcus aureus, Lactococcus lactis and Lactobacillus casei and 6-phospho-βglucosidase B of Escherichia coli build a subfamily inside a greater enzyme family, named the glycosal hydrolase family 1, which, hi addition, contains nine β-glycosidases of different origins. Kinetic and immunological evidence is provided in this report which strengthens the relationship of the four 6-phospho-β-glycosidases. It is shown that the 6-phospho-β-galactosidases and 6-phospho-β-glucosidase B are able to split aromatic β-galactoside phosphates and β-glucoside phosphates. The turnover numbers of hydrolysis of substrates with different epimerization at C-4 of the glycon vary up to 15-fold only. Two polydonal antisera, one derived against the native 6-phospho-β-galactosidase from S.aureus and the other derived against the 6-phospho-β-glucosidase B, cross-reacted with both enzymes. Peptides of the proteins were separated by reverse phase HPLC. The cross-reacting peptides were sequenced and shown to be localized at almost the same position in the aligned primary structures of both enzymes. An insertion of nine amino adds near these antigenic domains is unique for the 6-phospho-β-glycosidases and missing within the sequences of the β-glycoside-specific members of the family. The lacG gene of a 6-phospho-β-galactosidase negative S.aureus mutant was doned into E.coli and sequenced. In the totally inactive mutant protein only the glycine at position 332 was changed to an arginine. This amino acid is part of the sequence insertion near the antigenic domain reacting with both antisera. These data support the assumption that the region is of great importance for the function of the enzymes and that it is possible it determines the specificity of the phosphorylated form of the substrates. In addition, the 6-phospho-β-galactosidase of S.aureus was modified by sitedirected mutagenesis of the corresponding lacG gene hi order to replace residues Glul60 and Glu375, which were suspected of being involved hi the general acid catalysis of substrate hydrolysis, with glutamine residues. The mutant protein 160EQ retained some catalytic activity while the protein 375EQ was totally inactive. Glu375 is the active site nudeophile of the 6-phospho-β-galactosidase of S.aureus. It is located in the sequence motif ENG where Glu358 was identified as the catalytkally active nudeophile hi the β-glucosidase of Agrobacterium.