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J Biosci Bioeng. 2018 Feb;125(2):168-174. doi: 10.1016/j.jbiosc.2017.08.014. Epub 2017 Sep 11.

Structural modeling and mutagenesis of endo-β-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.

Author information

1
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China; Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan.
2
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
3
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan.
4
Tokyo R&D Center, Altif Laboratories, Inc., Tokyo 135-0064, Japan.
5
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan. Electronic address: y-chiba@aist.go.jp.

Abstract

Endo-β-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-β-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.

KEYWORDS:

Endo-Om; Endo-β-N-acetylglucosaminidase; Glycosidase family 85; N-glycan; Ogataea minuta

PMID:
28903882
DOI:
10.1016/j.jbiosc.2017.08.014
[Indexed for MEDLINE]

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