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Identification of a Catalytic Nucleophile-Activating Network in the endo - α -N- Acetylgalactosaminidase of Family GH101

Lønstrup Hansen, Anders ; M. Koivisto, Johanna ; Simonsen, Signe ; Dong, Zehui LU ; Crehuet, Ramon ; K. Hansen, Dennis and Willemoës, Martin (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)
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author
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publishing date
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}},
}