Mechanistic analysis of the blood group antigen-cleaving endo-beta-galactosidase from Clostridium perfringens.

The A and B antigens are of vital importance in blood transfusion and organ transplantation. The specificity of EABase, an endo-beta-galactosidase from C. perfringens, toward the cleavage of A and B trisaccharides from glycoconjugates is unique and holds significant potential for use in modifying blood group antigens on cell surfaces. The mechanism of this enzyme and others in its family (GH98) and the identities of its catalytic residues have not previously been experimentally determined. Direct 1H NMR analysis of the hydrolysis of a synthetic substrate, DNP-beta-A-trisaccharide, by EABase revealed that EABase is an inverting endo-beta-galactosidase. Both activated and nonactivated substrates were used to kinetically characterize EABase and its mutants (E354A, D429A, D453A, E467A, and E506A) at pH 6.0, 37 degrees C. Hydrolysis of DNP-beta-A-trisaccharide by EABase follows normal Michaelis-Menten kinetics with an apparent KM of 64 +/- 3 microM and a k(cat) of 105 +/- 5 min(-1). Mutation of two putative active site residues, D453 and E506, to alanine resulted in complete loss of activity, strongly suggesting that one or both of these residues functions as the base catalyst. The kinetic data also strongly suggest that E354 is the acid catalyst since the activity of the E354A mutant with nonactivated natural substrates is 1100-fold lower than that of the wild type enzyme, while its activity is only 10-fold lower when assayed with an activated aryl glycoside substrate (DNP-beta-A-trisaccharide). Further support is obtained through comparison of pH profiles for the wild type and E354A mutants: mutation of the acid catalyst eliminates the basic limb from the bell-shaped pH-dependence of k(cat)/KM seen for the wild type enzyme.

Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis.

The therapeutic potential of glycosides has made them an attractive target for drug development. The biological extraction and chemical synthesis of these molecules is often challenging and low yielding, thus alternative methods for the synthesis of polysaccharides are being pursued. A new class of enzymes, glycosynthases, which are nucleophile mutants of glycosidases, can perform the transglycosylation reaction without hydrolyzing the product, and thus provide a valuable resource for polysaccharide and glycan synthesis. Directed evolution of glycosynthases has expanded the repertoire of glycosidic linkages formed and the donors and acceptors (both sugar and nonsugar) that can be used by the glycosynthase. The application of new screening methods, such as FACS, to the directed evolution of glycosynthases will aid in the development of enzymes that are able to efficiently synthesize new, and therapeutically relevant glycosidic linkages.

Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis.

The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.