Characterization of a novel endo-beta-galactosidase specific for releasing the disaccharide GlcNAc alpha 1-->4Gal from glycoconjugates.
In contrast to the beta-linked GlcNAc, the alpha-linked GlcNAc has not been commonly found in glycoconjugates. We have recently revealed the presence of an unusual endo-beta-galactosidase (Endo-beta-Gal(GnGa)) in Clostridium perfringens capable of releasing GlcNAcalpha1-->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-beta-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-beta-Gal(GnGa) from 1 L of Escherichia coli DH5alpha culture, which was 170 times higher than that produced by the original clostridial strain. Endo-beta-Gal(GnGa) shares a low but significant sequence similarity with two other endo-beta-galactosidases (16-21% amino acid identity). It also shows some similarity with bacterial 1,3-1,4-beta-glucan 4-glucanohydrolases of the glycoside hydrolase family 16. Endo-beta-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-beta-Gal(GnGa) activity. By NMR spectroscopy, Endo-beta-Gal(GnGa) was found to act as a retaining enzyme.
Ashida
Hisashi
H
Department of Biochemistry, Tulane University Health Sciences Center School of Medicine, New Orleans, Louisiana 70112, USA.
Maskos
Karol
K
Li
Su-Chen
SC
Li
Yu-Teh
YT
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006001:Glycoconjugates / Q000378:metabolism*; D006026:Glycoside Hydrolases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004187:Disaccharides; D006001:Glycoconjugates; C033878:N-acetylgalactosaminyl-(1-4)-galactose; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase