Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme.
Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the transglycosylation activity.
Fujita
Kiyotaka
K
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan. kfujita@lif.kyoto-u.ac.jp
Kobayashi
Kazuo
K
Iwamatsu
Akihiro
A
Takeuchi
Makoto
M
Kumagai
Hidehiko
H
Yamamoto
Kenji
K
D000595:Amino Acid Sequence; D002384:Catalysis; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008358:Mannose / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000235:genetics*; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D006027:Glycosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010447:Peptide Hydrolases; D008358:Mannose