| 1. |
- Hansen, Dennis K., et al.
(författare)
-
Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galβ1-3GalNAc reactivity on Fetuin
- 2022
-
Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 725
-
Tidskriftsartikel (refereegranskat)abstract
- Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galβ1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galβ1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004–2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galβ1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galβ1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galβ1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galβ1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.
|
|
| 2. |
- Shuoker, Bashar, et al.
(författare)
-
Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria
- 2023
-
Ingår i: Nature Communications. - 2041-1723. ; 14
-
Tidskriftsartikel (refereegranskat)abstract
- The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.
|
|
| 3. |
- Teze, David, et al.
(författare)
-
The catalytic acid-base in GH109 resides in a conserved GGHGG loop and allows for comparable α-retaining and β-inverting activity in an N-acetylgalactosaminidase from Akkermansia muciniphila
- 2019
-
Ingår i: ChemRxiv. - : American Chemical Society (ACS).
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The study describes the first glycoside hydrolase that exhibits comparable levels of activity on α- and β-linked saccharide substrates. This enzyme, assigned into GH109, is encoded by the genome of the human gut symbiont Akkermansia muciniphila that is a model primary degrader of the heavily O-glycosylated mucin glycoprotein that coats the epithelial enterocytes.The elusive catalytic acid/base catalyst in GH109 enzymes is identified as a histidine that is presented by a flexible loop that positions it for catalysis on both α- and β-substrates. This dual activity may be an evolutionary adaptation to extend the range of substrates targeted by a single non-canonical NAD+-dependant GH.
|
|
| 4. |
- Teze, David, et al.
(författare)
-
The Catalytic Acid-Base in GH109 Resides in a Conserved GGHGG Loop and Allows for Comparable α-Retaining and β-Inverting Activity in an N-Acetylgalactosaminidase from Akkermansia muciniphila
- 2020
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 10:6, s. 3809-3819
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes active on glycosidic bonds are defined according to the stereochemistry of both substrates and products of the reactions they catalyze. The CAZy classification further assigns these enzymes into sequence-based families sharing a common stereochemistry for substrates (either α- or β-) and products (i.e., inverting or retaining mechanism). Here we describe the N-acetylgalactosaminidases AmGH109A and AmGH109B (i.e., GH109: glycoside hydrolase family 109) from the human gut symbiont Akkermansia muciniphila. Notably, AmGH109A displays α-retaining and β-inverting N-acetylgalactosaminidase activities with comparable efficiencies on natural disaccharides. This dual specificity could provide an advantage in targeting a broader range of host-derived glycans. We rationalize this discovery through bioinformatics, structural, mutational, and computational studies, unveiling a histidine residing in a conserved GGHGG motif as the elusive catalytic acid-base of the GH109 family.
|
|
