Characterization of a Novel Endo-β-galactosidase Specific for Releasing the Disaccharide GlcNAcα1→4Gal from Glycoconjugates^†,‡

In contrast to the β-linked GlcNAc, the α-linked GlcNAc has not been commonly found in glycoconjugates. We have recently revealed the presence of an unusual endo-β-galactosidase (Endo-β-Gal_GnGa) in Clostridium perfringens capable of releasing GlcNAcα1→4Gal from glycans expressed in the gastric mucous cell-type mucin [Ashida, H., Anderson, K., Nakayama, J., Maskos, K., Chou, C.-W., Cole, R. B., Li, S.-C., and Li, Y.-T. (2001) J. Biol. Chem. 276, 28226−28232]. To characterize Endo-β-Gal_GnGa, we have cloned its gene, gngC, from the genomic DNA library prepared from C. perfringens ATCC10543. The gene encodes 420 amino acid residues including a 17-residue signal peptide at the N-terminus. Using pUC18, we were able to prepare 25 mg of the fully active and pure recombinant Endo-β-Gal_GnGa from 1 L of Escherichia coli DH5α culture, which was 170 times higher than that produced by the original clostridial strain. Endo-β-Gal_GnGa shares a low but significant sequence similarity with two other endo-β-galactosidases (16−21% amino acid identity). It also shows some similarity with bacterial 1,3-1,4-β-glucan 4-glucanohydrolases of the glycoside hydrolase family 16. Endo-β-Gal_GnGa was found to contain the EXDX(X)E sequence (Glu-168 to Glu-173), that has been identified as the catalytic motif of families 16 and 7 retaining glycoside hydrolases. We have used site-directed mutagenesis to show that Glu-168 and Glu-173 were essential for the Endo-β-Gal_GnGa activity. By NMR spectroscopy, Endo-β-Gal_GnGa was found to act as a retaining enzyme.