In the 2016/2017 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the VP1 gene and RdRp region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains VP1 gene and RdRp region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the VP1 gene and RdRp region were around 10-3 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the VP1 gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.

Endo-␤-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the ␤1,4 linkage of N,N-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glycoengineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the sitedirected mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K m value of Y217F for an acceptor substrate 4-methylumbelliferyl-␤-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn 175 "knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-␤-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.Endo-␤-N-acetylglucosaminidase (EC 3.2.1.96) (ENGase) 3 catalyzes hydrolysis of the ␤1,4-glycosidic linkage of the N,NЈ-diacetylchitobiose moiety in the core of asparagine-linked glycan of various glycoproteins and glycopeptides. This type of enzyme is widely distributed in animals, plants, fungi, and bacteria. Several bacterial enzymes, such as Endo-H from Streptomyces plicatus (1) and Endo-F 1 from Flavobacterium meningosepticum (2), were cloned and classified into glycoside hydrolase (GH) family 18 in the CAZy data base (available on the World Wide Web), which may share a common evolutional origin with GH18 chitinases. The other ENGases are distinct from the enzymes of the GH18 chitinase family and are classified into the GH family 85. We and others have previously reported that several ENGases of the GH85 family showed significant transglycosylation activity (i.e. the ability to transfer the releasing glycan to an acceptor other than water to form a new glycosidic linkage) (3-6). These ENGases include Endo-M from Mucor hiemalis (3), Endo-A from Arthrobactor protophormiae (4), Endo-CE from Caenorhabditi...

We found endo-␣-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl ␤1,3-N-acetyl-Dgalactosamine (Gal␤1,3GalNAc) ␣-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590 -1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Gal␤1,3GalNAc disaccharide from Gal␤1,3GalNAc␣1pNP and asialofetuin, but did not release GalNAc, Gal␤1,3(GlcNAc␤1,6)GalNAc, GlcNAc␤1,3GalNAc, and Gal␤1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by 1 H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono-and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-␣-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).Mucin-type oligosaccharides (O-glycans) are involved in several important biological events including cell-to-cell communication in higher eukaryotes, bacterial adhesion to host cells, and so on (1). Eight core-type structures of O-glycans have been proposed, and among them, galactosyl ␤1,3-N-acetyl-D-galactosamine (Gal␤1,3GalNAc) 2 attached to the serine or threonine residue via an ␣-linkage (Core 1, also referred to as T antigen) is one of the most abundant core structures found in mucin glycoproteins that are widely distributed in intestinal tracts of human and animals. This immunogenic structure is normally masked by further glycosylation such as sialylation (sialyl T antigen) and N-acetylglucosaminylation (Core 2 structure). However, it has been shown that T antigen is frequently exposed on the cell surface of carcinoma and T-cell lymphoma, and this unmasked form is thought to be involved in tumor cell adhesion and tissue invasion (2, 3). In addition, incomplete synthesis of the Core 1 structure associates with several autoimmune diseases including IgA nephropathy (4), Tn-syndrome (5), and Henoch-Schönlein purpura (6).Endo-␣-N-acetylgalactosaminidase (EC 3.2.1.97; endo-␣-GalNAcase, glycopeptide ␣-N-acetylgalactosaminidase) is a unique enzyme that hydrolyzes Core 1-type O-glycan from glycoproteins. This enzyme also serves as a powerful tool for elucidating the presence of O-glycan...

Background: ␤-L-Arabinofuranosyl linkages are found in many plant biopolymers, but the degradation enzyme has never been found. Results: A novel ␤-L-arabinofuranosidase was found in Bifidobacterium longum. Conclusion: ␤-L-Arabinofuranosidase plays a key role in Bifidobacterium longum for ␤-L-arabinooligosaccharide usage. Significance: The members of the DUF1680 family might be used for the degradation of plant biopolymers.

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Extensin is a glycoprotein that is rich in hydroxyprolines linked to ␤-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel ␤-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38 -98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-␤1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-␤1,2-Araf-␤1,2-Araf-␤-Hyp (Ara 3 -Hyp) but not Araf-␣1,3-Araf-␤1,2-Araf-␤1,2-Araf-␤-Hyp (Ara 4 -Hyp) or Araf-␤1,2-Araf-␤-Hyp (Ara 2 -Hyp), which indicated that it was specific for unmodified Ara 3 -Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyplinked ␤-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.

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We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.