Mutational engineering, growth selection, expression, purification and crystallization of ligand bound endo-beta-N-acetylglucosaminidase T (Endo-T) from Hypocrea jecorina
Recently, one of the first fungal-expressed, deglycosylating, Endo-beta-N-acetylglucosaminidases was found in the extracellular medium of soft-rot ascomycete Hypocrea jecorina (a.k.a. Trichoderma reesei) belonging to glycoside hydrolase family 18 (GH18). It was named Endo-T and has been shown to pos...
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| Formato: | H1 |
| Lenguaje: | Inglés |
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SLU/Dept. of Molecular Biology (until 131231)
2010
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| Materias: |
| _version_ | 1855570305763770368 |
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| author | Digre, Andreas |
| author_browse | Digre, Andreas |
| author_facet | Digre, Andreas |
| author_sort | Digre, Andreas |
| collection | Epsilon Archive for Student Projects |
| description | Recently, one of the first fungal-expressed, deglycosylating, Endo-beta-N-acetylglucosaminidases was found in the extracellular medium of soft-rot ascomycete Hypocrea jecorina (a.k.a. Trichoderma reesei) belonging to glycoside hydrolase family 18 (GH18). It was named Endo-T and has been shown to possess similar substrate specificities as Endo-H from Streptomyces plicatus, a deglycosylating enzyme, frequently used in the field of glycoproteomics. In this study an oligomannosidic N-glycan was introduced in the active site of crystallized Endo-T under acidic pH conditions below pH 3. The ligand-containing crystal diffracted on a synchrotron x-ray source to a resolution of 1.65Å. The resulting Endo-T structure was found to contain a bound ligand consisting of a hydrolyzed Manα1-6(Manα1-3)Manα1-6Manβ1-4GlcNAc N-glycan, lacking the aspargine linked N-acetylglucosamine residue of N-glycans. Furthermore, electron density was missing for several of the distal glycone mannose residues. The anomeric carbon of the distal N-acetylglucosamine residue was found to be positioned 6.88Å from the proposed catalytic amino acid residue, Glu131, indicating a descending motion during hydrolysis. An unidentified electron density was found after the last structure refinement, apparently shaped as a furanose ring, next to the N-glycan ligand in the active site around unit 8 of the (β/α)8-barrel. Proximal substrate positioning and the loop-structure of Endo-T suggests aglycone docking centered over (β/α)8-barrel unit 5. |
| format | H1 |
| id | RepoSLU1432 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés |
| publishDate | 2010 |
| publishDateSort | 2010 |
| publisher | SLU/Dept. of Molecular Biology (until 131231) |
| publisherStr | SLU/Dept. of Molecular Biology (until 131231) |
| record_format | eprints |
| spelling | RepoSLU14322012-04-20T14:14:07Z Mutational engineering, growth selection, expression, purification and crystallization of ligand bound endo-beta-N-acetylglucosaminidase T (Endo-T) from Hypocrea jecorina Digre, Andreas Endo-T Endo-beta-N-acetylglucosaminidase Deglycosylation EC 3.2.1.96 Crystallography Ligand bound structure High mannose N-glycan HPAEC-PAD Recently, one of the first fungal-expressed, deglycosylating, Endo-beta-N-acetylglucosaminidases was found in the extracellular medium of soft-rot ascomycete Hypocrea jecorina (a.k.a. Trichoderma reesei) belonging to glycoside hydrolase family 18 (GH18). It was named Endo-T and has been shown to possess similar substrate specificities as Endo-H from Streptomyces plicatus, a deglycosylating enzyme, frequently used in the field of glycoproteomics. In this study an oligomannosidic N-glycan was introduced in the active site of crystallized Endo-T under acidic pH conditions below pH 3. The ligand-containing crystal diffracted on a synchrotron x-ray source to a resolution of 1.65Å. The resulting Endo-T structure was found to contain a bound ligand consisting of a hydrolyzed Manα1-6(Manα1-3)Manα1-6Manβ1-4GlcNAc N-glycan, lacking the aspargine linked N-acetylglucosamine residue of N-glycans. Furthermore, electron density was missing for several of the distal glycone mannose residues. The anomeric carbon of the distal N-acetylglucosamine residue was found to be positioned 6.88Å from the proposed catalytic amino acid residue, Glu131, indicating a descending motion during hydrolysis. An unidentified electron density was found after the last structure refinement, apparently shaped as a furanose ring, next to the N-glycan ligand in the active site around unit 8 of the (β/α)8-barrel. Proximal substrate positioning and the loop-structure of Endo-T suggests aglycone docking centered over (β/α)8-barrel unit 5. SLU/Dept. of Molecular Biology (until 131231) 2010 H1 eng https://stud.epsilon.slu.se/1432/ |
| spellingShingle | Endo-T Endo-beta-N-acetylglucosaminidase Deglycosylation EC 3.2.1.96 Crystallography Ligand bound structure High mannose N-glycan HPAEC-PAD Digre, Andreas Mutational engineering, growth selection, expression, purification and crystallization of ligand bound endo-beta-N-acetylglucosaminidase T (Endo-T) from Hypocrea jecorina |
| title | Mutational engineering, growth selection, expression, purification and crystallization of ligand bound
endo-beta-N-acetylglucosaminidase T (Endo-T)
from Hypocrea jecorina |
| title_full | Mutational engineering, growth selection, expression, purification and crystallization of ligand bound
endo-beta-N-acetylglucosaminidase T (Endo-T)
from Hypocrea jecorina |
| title_fullStr | Mutational engineering, growth selection, expression, purification and crystallization of ligand bound
endo-beta-N-acetylglucosaminidase T (Endo-T)
from Hypocrea jecorina |
| title_full_unstemmed | Mutational engineering, growth selection, expression, purification and crystallization of ligand bound
endo-beta-N-acetylglucosaminidase T (Endo-T)
from Hypocrea jecorina |
| title_short | Mutational engineering, growth selection, expression, purification and crystallization of ligand bound
endo-beta-N-acetylglucosaminidase T (Endo-T)
from Hypocrea jecorina |
| title_sort | mutational engineering, growth selection, expression, purification and crystallization of ligand bound
endo-beta-n-acetylglucosaminidase t (endo-t)
from hypocrea jecorina |
| topic | Endo-T Endo-beta-N-acetylglucosaminidase Deglycosylation EC 3.2.1.96 Crystallography Ligand bound structure High mannose N-glycan HPAEC-PAD |