THE 3-DIMENSIONAL STRUCTURE OF 6-PHOSPHO-BETA-GALACTOSIDASE FROM LACTOCOCCUS-LACTIS

Background: The enzyme 6-phospho-beta-galactosidase hydrolyzes phospho lactose, the product of a phosphoenolpyruvate-dependent phosphotransfe rase system. It belongs to glycosidase family 1 and no structure has y et been published for a member of this family. Results: The crystal st ructure of 6-phospho-beta-galactosidase was determined at 2.3 Angstrom resolution by multiple isomorphous replacement, using the wild-type e nzyme and a designed cysteine mutant. A second crystal form, found wit h the mutant enzyme, was solved by molecular replacement, yielding the conformation of two chain loops that are invisible in the first cryst al form. The active center, located through catalytic residues identif ied in previous studies, cannot be accessed by the substrate if the tw o loops are in their defined conformation. The enzyme contains a (beta alpha)(8) barrel and the relationship of its chain fold to that of ot her glycosidases has been quantified. As a side issue, we observed tha t a cysteine point mutant designed for X-ray analysis crystallized mai nly as a symmetric dimer around an intermolecular disulfide bridge for med by the newly introduced cysteine. Conclusions: The presented analy sis provides a basis on which to model all other family 1 members and thereby will help in elucidating the catalytic mechanism of these sequ ence-related enzymes. Moreover, this enzyme belongs to a superfamily o f glycosidases sharing a (beta alpha)(8) barrel with catalytic glutama tes/aspartates at the ends of the fourth and the seventh strands of th e beta barrel.