Identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica S66T

Triinu Visnapuu; David Teze; Christian Kjeldsen; Aleksander Lie; Jens Øllgaard Duus; Corinne André-Miral; Lars Haastrup Pedersen; Peter Stougaard; Birte Svensson

doi:10.3390/ijms21020417

Identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica S66^T

Triinu Visnapuu^*, David Teze, Christian Kjeldsen, Aleksander Lie, Jens Øllgaard Duus, Corinne André-Miral, Lars Haastrup Pedersen, Peter Stougaard, Birte Svensson

^*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

Abstract

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.

Originalsprog	Engelsk
Artikelnummer	417
Tidsskrift	International Journal of Molecular Sciences
Vol/bind	21
Nummer	2
ISSN	1661-6596
DOI	https://doi.org/10.3390/ijms21020417
Status	Udgivet - 2 jan. 2020
Udgivet eksternt	Ja

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10.3390/ijms21020417

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http://www.scopus.com/inward/record.url?scp=85077856787&partnerID=8YFLogxK

Citationsformater

Visnapuu, T., Teze, D., Kjeldsen, C., Lie, A., Duus, J. Ø., André-Miral, C., Pedersen, L. H., Stougaard, P., & Svensson, B. (2020). Identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica S66^T. International Journal of Molecular Sciences , 21(2), Artikel 417. https://doi.org/10.3390/ijms21020417

@article{bc9534a185494777aae527d138c0cbe6,

title = "Identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica S66T",

abstract = "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 S66T, 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: kcat /KM 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.",

keywords = "Acceptor diversity, GH20, Glycoside hydrolase, Human milk oligosaccharides, Lacto-N-triose II, N-acetylhexosamine specificity, NAG-oxazoline, NMR, Phylogenetic analysis, Transglycosylation",

author = "Triinu Visnapuu and David Teze and Christian Kjeldsen and Aleksander Lie and Duus, {Jens {\O}llgaard} and Corinne Andr{\'e}-Miral and Pedersen, {Lars Haastrup} and Peter Stougaard and Birte Svensson",

year = "2020",

month = jan,

day = "2",

doi = "10.3390/ijms21020417",

language = "English",

volume = "21",

journal = "International Journal of Molecular Sciences ",

issn = "1661-6596",

publisher = "MDPI AG",

number = "2",

}

Visnapuu, T, Teze, D, Kjeldsen, C, Lie, A, Duus, JØ, André-Miral, C, Pedersen, LH, Stougaard, P & Svensson, B 2020, 'Identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica S66^T', International Journal of Molecular Sciences , bind 21, nr. 2, 417. https://doi.org/10.3390/ijms21020417

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 et al.
I: International Journal of Molecular Sciences , Bind 21, Nr. 2, 417, 02.01.2020.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

TY - JOUR

T1 - Identification and characterization of a β-n-acetylhexosaminidase with a biosynthetic activity from the marine bacterium paraglaciecola hydrolytica S66T

AU - Visnapuu, Triinu

AU - Teze, David

AU - Kjeldsen, Christian

AU - Lie, Aleksander

AU - Duus, Jens Øllgaard

AU - André-Miral, Corinne

AU - Pedersen, Lars Haastrup

AU - Stougaard, Peter

AU - Svensson, Birte

PY - 2020/1/2

Y1 - 2020/1/2

N2 - 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 S66T, 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: kcat /KM 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.

AB - 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 S66T, 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: kcat /KM 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.

KW - Acceptor diversity

KW - GH20

KW - Glycoside hydrolase

KW - Human milk oligosaccharides

KW - Lacto-N-triose II

KW - N-acetylhexosamine specificity

KW - NAG-oxazoline

KW - NMR

KW - Phylogenetic analysis

KW - Transglycosylation

UR - http://www.scopus.com/inward/record.url?scp=85077856787&partnerID=8YFLogxK

U2 - 10.3390/ijms21020417

DO - 10.3390/ijms21020417

M3 - Journal article

C2 - 31936522

AN - SCOPUS:85077856787

SN - 1661-6596

VL - 21

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

IS - 2

M1 - 417

ER -