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.
Highlights
A new marine bacterial species Paraglaciecola hydrolytica S66T of the family Alteromonadaceae isolated from eelgrass (Zostera sp.) was shown by genome-sequencing [1] to encode 270 protein modules potentially acting on carbohydrates, 188 of which belong to enzyme families involved in degradation of carbohydrates [2,3]
P. hydrolytica was grown in marine mineral medium supplemented with a mixture of chitooligosaccharides (GlcNAc)1–6 as the sole carbon source, which were hydrolyzed to glycosidic bonds with the release of N-acetylglucosamine (GlcNAc) (Supplementary Information 1, Figure S1A,B)
P. hydrolytica, did not hydrolyze α-chitin from crab shells used to supplement the marine mineral medium, as neither GlcNAc nor chitooligosaccharides appeared during the incubation (Figure S1C). β-NAHA activity from P. hydrolytica was detected by a hydrolysis of the chromogenic 5-bromo-4-chloro-3-indolyl N-acetyl-β-d-glucosaminide (X-GlcNAc) on a complex marine agar medium (Figure S1D)
Summary
A new marine bacterial species Paraglaciecola hydrolytica S66T of the family Alteromonadaceae isolated from eelgrass (Zostera sp.) was shown by genome-sequencing [1] to encode 270 protein modules potentially acting on carbohydrates, 188 of which belong to enzyme families involved in degradation of carbohydrates [2,3]. The large number of encoded carbohydrate-active enzymes (CAZymes) [4] and the flexibility with regard to carbon source indicates a very promising potential of the genome of P. hydrolytica for the discovery of enzymes with rare or not yet described activities. Human milk oligosaccharides (HMOs) in particular are considered beneficial and needed for research and clinical trials within nutrition and as ingredients in functional foods and infant formulas [6,7,8]. The chemical and enzymatic production of HMOs and their precursors or purification from natural sources are problematical [6,11,12], which creates bottlenecks for assessing the functional roles and applications of HMOs [13,14,15]
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