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Protein Eng. 1993 Nov;6(8):913-20.

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 lacG gene of Staphylococcus aureus.

Author information

1
Abteilung für Biologie, Ruhr-Universität Bochum, Germany.

Abstract

The 6-phospho-beta-galactosidase of Staphylococcus aureus, Lactococcus lactis and Lactobacillus casei and 6-phospho-beta-glucosidase B of Escherichia coli build a subfamily inside a greater enzyme family, named the glycosal hydrolase family 1, which, in addition, contains nine beta-glycosidases of different origins. Kinetic and immunological evidence is provided in this report which strengthens the relationship of the four 6-phospho-beta-glycosidases. It is shown that the 6-phospho-beta-galactosidases and 6-phospho-beta-glucosidase B are able to split aromatic beta-galactoside phosphates and beta-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 polyclonal antisera, 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 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 acids near these antigenic domains is unique for the 6-phospho-beta-glycosidases and missing within the sequences of the beta-glycoside-specific members 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 changed to an arginine. This amino acid is part of the sequence insertion near the antigenic domain reacting with both antisera.(ABSTRACT TRUNCATED AT 250 WORDS).

PMID:
8309940
DOI:
10.1093/protein/6.8.913
[Indexed for MEDLINE]

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