6-PHOSPHO-BETA-GALACTOSIDASES OF GRAM-POSITIVE AND 6-PHOSPHO-BETA-GLUCOSIDASE-B OF GRAM-NEGATIVE BACTERIA - COMPARISON OF STRUCTURE AND FUNCTION BY KINETIC AND IMMUNOLOGICAL METHODS AND MUTAGENESIS OF THE LACGGENE OF STAPHYLOCOCCUS-AUREUS

The 6-phospho-beta-galactosidase of Staphylococcus aureus, Lactococcus lactis and Lactobacillus casei and 6-phospho-beta-glucosidase B of Es cherichia coli build a subfamily inside a greater enzyme family, named the glycosal hydrolase family 1, which, in addition, contains nine be ta-glycosidases of different origins. Kinetic and immunological eviden ce is provided in this report which strengthens the relationship of th e four 6-phospho-beta-glycosidases. It is shown that the 6-phospho-bet a-galactosidases and 6-phospho-beta-glucosidase B are able to split ar omatic beta-galactoside phosphates and beta-glucoside phosphates. The turnover numbers of hydrolysis of substrates with different epimerizat ion at C-4 of the glycon vary up to 15-fold only. Two polyclonal antis era, one derived against the native 6-phospho-beta-galactosidase from S.aureus and the other derived against the 6-phospho-beta-glucosidase B, cross-reacted with both enzymes. Peptides of the proteins were sepa rated by reverse phase HPLC. The cross-reacting peptides were sequence d and shown to be localized at almost the same position in the aligned primary structures of both enzymes. An insertion of nine amino acids near these antigenic domains is unique for the 6-phospho-beta-glycosid ases and missing within the sequences of the beta-glycoside-specific m embers of the family. The lacG gene of a 6-phospho-beta-galactosidase negative S.aureus mutant was cloned into E.coli and sequenced. In the totally inactive mutant protein only the glycine at position 332 was c hanged to an arginine. This amino acid is part of the sequence inserti on near the antigenic domain reacting with both antisera. These data s upport the assumption that the region is of great importance for the f unction of the enzymes and that it is possible it determines the speci ficity of the phosphorylated form of the substrates. In addition, the 6-phospho-beta-galactosidase of S.aureus was modified by site-directed mutagenesis of the corresponding lacG gene in order to replace residu es Glu160 and Glu375, which were suspected of being involved in the ge neral acid catalysis of substrate hydrolysis, with glutamine residues. The mutant protein 160EQ retained some catalytic activity while the p rotein 375EQ was totally inactive. Glu375 is the active site nucleophi le of the 6-phospho-beta-galactosidase of S.aureus. It is located in t he sequence motif ENG where Glu358 was identified as the catalytically active nucleophile in the beta-glucosidase of Agrobacterium.