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

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  • Triinu Visnapuu, Danmarks Tekniske Universitet, University Tartu
  • ,
  • David Teze, Danmarks Tekniske Universitet
  • ,
  • Christian Kjeldsen, Danmarks Tekniske Universitet
  • ,
  • Aleksander Lie, Aalborg Universitet, Kebony Norge AS
  • ,
  • Jens Øllgaard Duus, Danmarks Tekniske Universitet
  • ,
  • Corinne André-Miral, Universite de Nantes
  • ,
  • Lars Haastrup Pedersen, Department of Chemistry and Bioscience, Aalborg Universitet
  • ,
  • Peter Stougaard
  • Birte Svensson, Danmarks Tekniske Universitet

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 50C, 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.

OriginalsprogEngelsk
Artikelnummer417
TidsskriftInternational Journal of Molecular Sciences
Vol/bind21
Nummer2
ISSN1661-6596
DOI
StatusUdgivet - 2 jan. 2020
Eksternt udgivetJa

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