Identification of a Catalytic Nucleophile-Activating Network in the endo - α -N- Acetylgalactosaminidase of Family GH101
(2021) In Biochemistry- Abstract
- Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galβ1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N... (More)
- Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galβ1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galβ(1–3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/02221da3-5263-4c24-81c9-f7337204236e
- author
- Lønstrup Hansen, Anders ; M. Koivisto, Johanna ; Simonsen, Signe ; Dong, Zehui LU ; Crehuet, Ramon ; K. Hansen, Dennis and Willemoës, Martin
- publishing date
- 2021-10-25
- type
- Contribution to journal
- publication status
- published
- in
- Biochemistry
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:85118763364
- ISSN
- 0006-2960
- DOI
- 10.1021/acs.biochem.1c00596
- language
- English
- LU publication?
- no
- id
- 02221da3-5263-4c24-81c9-f7337204236e
- date added to LUP
- 2022-07-04 12:47:12
- date last changed
- 2023-04-24 04:01:34
@article{02221da3-5263-4c24-81c9-f7337204236e, abstract = {{Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galβ1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galβ(1–3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.}}, author = {{Lønstrup Hansen, Anders and M. Koivisto, Johanna and Simonsen, Signe and Dong, Zehui and Crehuet, Ramon and K. Hansen, Dennis and Willemoës, Martin}}, issn = {{0006-2960}}, language = {{eng}}, month = {{10}}, publisher = {{The American Chemical Society (ACS)}}, series = {{Biochemistry}}, title = {{Identification of a Catalytic Nucleophile-Activating Network in the endo - α -N- Acetylgalactosaminidase of Family GH101}}, url = {{http://dx.doi.org/10.1021/acs.biochem.1c00596}}, doi = {{10.1021/acs.biochem.1c00596}}, year = {{2021}}, }