Cargando…
Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T)
β-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydroly...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014002/ https://www.ncbi.nlm.nih.gov/pubmed/31936522 http://dx.doi.org/10.3390/ijms21020417 |
_version_ | 1783496528422240256 |
---|---|
author | Visnapuu, Triinu Teze, David Kjeldsen, Christian Lie, Aleksander Duus, Jens Øllgaard André-Miral, Corinne Pedersen, Lars Haastrup Stougaard, Peter Svensson, Birte |
author_facet | Visnapuu, Triinu Teze, David Kjeldsen, Christian Lie, Aleksander Duus, Jens Øllgaard André-Miral, Corinne Pedersen, Lars Haastrup Stougaard, Peter Svensson, Birte |
author_sort | Visnapuu, Triinu |
collection | PubMed |
description | β-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66(T), are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied β-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc)(2) and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: k(cat)/K(M) being 341 and 344 mM(−1)·s(−1), respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2- or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases. |
format | Online Article Text |
id | pubmed-7014002 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-70140022020-03-09 Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) Visnapuu, Triinu Teze, David Kjeldsen, Christian Lie, Aleksander Duus, Jens Øllgaard André-Miral, Corinne Pedersen, Lars Haastrup Stougaard, Peter Svensson, Birte Int J Mol Sci Article β-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66(T), are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied β-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc)(2) and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: k(cat)/K(M) being 341 and 344 mM(−1)·s(−1), respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2- or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases. MDPI 2020-01-09 /pmc/articles/PMC7014002/ /pubmed/31936522 http://dx.doi.org/10.3390/ijms21020417 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Visnapuu, Triinu Teze, David Kjeldsen, Christian Lie, Aleksander Duus, Jens Øllgaard André-Miral, Corinne Pedersen, Lars Haastrup Stougaard, Peter Svensson, Birte Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) |
title | Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) |
title_full | Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) |
title_fullStr | Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) |
title_full_unstemmed | Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) |
title_short | Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66(T) |
title_sort | identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica s66(t) |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014002/ https://www.ncbi.nlm.nih.gov/pubmed/31936522 http://dx.doi.org/10.3390/ijms21020417 |
work_keys_str_mv | AT visnapuutriinu identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT tezedavid identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT kjeldsenchristian identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT liealeksander identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT duusjensøllgaard identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT andremiralcorinne identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT pedersenlarshaastrup identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT stougaardpeter identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t AT svenssonbirte identificationandcharacterizationofabnacetylhexosaminidasewithabiosyntheticactivityfromthemarinebacteriumparaglaciecolahydrolyticas66t |