Kazuhiro Chiku

Discovery of Two β-1,2-Mannoside Phosphorylases Showing Different Chain-Length Specificities from Thermoanaerobacter sp. X-514

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

Effects of Lipooligosaccharide Inner Core Truncation on Bile Resistance and Chick Colonization by Campylobacter jejuni

Campylobacter jejuni is the most common bacterium that causes diarrhea worldwide, and chickens are considered the main reservoir of this pathogen. This study investigated the effects of serial truncation of lipooligosaccharide (LOS), a major component of the outer membrane of C. jejuni, on its bile resistance and intestinal colonization ability in chickens. Genes encoding manno-heptose synthetases or glycosyltransferases were inactivated to generate isogenic mutants. Serial truncation of the LOS core oligosaccharide caused a stepwise increase in susceptibilities of two C. jejuni strains, NCTC 11168 and 81-176, to bile acids. Inactivation of hldE, hldD, or waaC caused severe truncation of the core oligosaccharide, which greatly increased the susceptibility to bile acids. Both wild-type strains grew normally in chicken intestinal extracts, whereas the mutants with severe oligosaccharide truncation were not detected 12 h after inoculation. These mutants attained viable bacterial counts in the bile acid-free extracts 24 h after inoculation. The wild-type strain 11-164 was present in the cecal contents at >107 CFU/g on 5 days after challenge infection and after this time period, whereas its hldD mutant was present at <103 CFU/g throughout the experimental period. Trans-complementation of the hldD mutant with the wild-type hldD allele completely restored the in vivo colonization level to that of the wild-type strain. Mutants with a shorter LOS had higher hydrophobicities. Thus, the length of the LOS core oligosaccharide affected the surface hydrophobicity and bile resistance of C. jejuni as well as its ability to colonize chicken intestines.

Thermal decomposition of β-D-galactopyranosyl-(1->3)-2-acetamido-2-deoxy-D-hexopyranoses under neutral conditions

beta-d-Galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-d-glucose (LNB) and beta-d-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-d-galactose (GNB) decompose rapidly upon heating into d-galactose and mono-dehydrated derivatives of the corresponding 2-acetamido-2-deoxy-d-hexoses, including 2-acetamido-2,3-dideoxy-hex-2-enofuranoses and bicyclic 2-acetamido-3,6-anhydro-2-deoxy-hexofuranoses. The decomposition is conducted under neutral conditions where glycosyl linkages are generally believed to be stable. The half-lives of LNB and GNB were 8.1min and 20min, respectively, at 90 degrees C and pH 7.5. The pH dependency of decomposition rates suggests that the instabilities are an extension of the conditions for the peeling reaction, often observed with glycans of O-linked glycoproteins under alkaline conditions. Such decomposition under the neutral conditions is commonly observed with 3-O-linked reducing aldoses.

Potassium ion‐dependent trehalose phosphorylase from halophilic Bacillus selenitireducens MLS10

We discovered a potassium ion‐dependent trehalose phosphorylase (Bsel_1207) belonging to glycoside hydrolase family 65 from halophilic Bacillus selenitireducens MLS10. Under high potassium ion concentrations, the recombinant Bsel_1207 produced in Escherichia coli existed as an active dimeric form that catalyzed the reversible phosphorolysis of trehalose in a typical sequential bi bi mechanism releasing β‐d‐glucose 1‐phosphate and d‐glucose. Decreasing potassium ion concentrations significantly reduced thermal and pH stabilities, leading to formation of inactive monomeric Bsel_1207.

An inverting β-1,2-mannosidase belonging to glycoside hydrolase family 130 from Dyadobacter fermentans.

The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of β‐d‐mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using α‐d‐mannose 1‐phosphate but it released α‐d‐mannose from β‐1,2‐mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a β‐1,2‐mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of β‐1,2‐mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the β‐d‐mannosidic bond.

Characterization and crystal structure determination of β-1,2-mannobiose phosphorylase from Listeria innocua

Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for β‐mannosides. Here, we characterized β‐1,2‐mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with β‐1,2‐linked mannooligosaccharides. β‐1,2‐Mannotriose was bound in a U‐shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open–close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

Comparative analysis of flagellin glycans among pathovars of phytopathogenic Pseudomonas syringae

Flagellin is a principal component of the flagellum filament. Previously, we reported that the flagellin of Pseudomonas syringae pv. tabaci 6605 (Pta6605) was glycosylated by oligosaccharides composed of two or three l-rhamnosyl (l-Rha) residues and a terminal 4,6-dideoxy-4-(3-hydroxybutanamide)-2-O-methylglucopyranosyl residue. In this study, we characterized the chemical structure of flagellin glycans in P. syringae pathovars glycinea race 4 (Pgl4), phaseolicola 1448A (Pph1448A), tomato DC3000 (PtoDC3000), and syringae B728a (PsyB728a). Flagellin glycans were released by hydrazinolysis, labeled on the reducing ends with 2-aminopyridine (PA), and the PA-labeled oligosaccharides were isolated by high-performance liquid chromatography. The purified PA-labeled glycans were analyzed by mass spectrometry and NMR spectroscopy. The results showed that the glycans on flagellin of Pgl4, PtoDC3000, and Pph1448A were identical to those of Pta6605, which were characterized previously. The flagellin of PsyB728a is O-glycosylated with a novel trisaccharide identified as 2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→2)-3-O-methyl-α-L-rhamnopyranosyl-(1→2)-L-rhamnose. Our data indicate that flagellin glycosylation of P. syringae pathovars has universality with little diversity.