Kiyotaka Fujita

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum FUNCTIONAL ELUCIDATION OF A DUF1680 FAMILY MEMBER

Background: β-l-Arabinofuranosyl linkages are found in many plant biopolymers, but the degradation enzyme has never been found. Results: A novel β-l-arabinofuranosidase was found in Bifidobacterium longum. Conclusion: β-l-Arabinofuranosidase plays a key role in Bifidobacterium longum for β-l-arabinooligosaccharides usage. Significance: The members of DUF1680 family might be used for the degradation of plant biopolymers. Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.

Syntheses of mucin-type O -glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of Bifidobacterium endo-α- N -acetylgalactosaminidase

Endo-α-N-acetylgalactosaminidase catalyzes the release of Galβ1-3GalNAc from the core 1-type O-glycan (Galβ1-3GalNAcα1-Ser/Thr) of mucin glycoproteins and synthetic p-nitrophenyl (pNP) α-linked substrates. Here, we report the enzymatic syntheses of core 1 disaccharide-containing glycopeptides using the transglycosylation activity of endo-α-N-acetylgalactosaminidase (EngBF) from Bifidobacterium longum. The enzyme directly transferred Galβ1-3GalNAc to serine or threonine residues of bioactive peptides such as PAMP-12, bradykinin, peptide-T and MUC1a when Galβ1-3GalNAcα1-pNP was used as a donor substrate. The enzyme was also found to catalyze the reverse-hydrolysis reaction. EngBF synthesized the core 1 disaccharide-containing oligosaccharides when the enzyme was incubated with either glucose or lactose and Galβ1-3GalNAc prepared from porcine gastric mucin using bifidobacterial cells expressing endo-α-N-acetylgalactosaminidase. Synthesized oligosaccharides are promising prebiotics for bifidobacteria.

Bifidobacterium longum subsp. longum Exo-β-1,3-Galactanase, an Enzyme for the Degradation of Type II Arabinogalactan

ABSTRACT Type II arabinogalactan (AG-II) is a suitable carbohydrate source for Bifidobacterium longum subsp. longum, but the degradative enzymes have never been characterized. In this study, we characterized an exo-β-1,3-galactanase, BLLJ_1840, belonging to glycoside hydrolase family 43 from B. longum subsp. longum JCM1217. The recombinant BLLJ_1840 expressed in Escherichia coli hydrolyzed β-1,3-linked galactooligosaccharides but not β-1,4- and β-1,6-linked galactooligosaccharides. The enzyme also hydrolyzed larch wood arabinogalactan (LWAG), which comprises a β-1,3-linked galactan backbone with β-1,6-linked galactan side chains. The k cat/Km ratio of dearabinosylated LWAG was 24-fold higher than that of β-1,3-galactan. BLLJ_1840 is a novel type of exo-β-1,3-galactanase with a higher affinity for the β-1,6-substituted β-1,3-galactan than for nonsubstituted β-1,3-galactan. BLLJ_1840 has 27% to 28% identities with other characterized exo-β-1,3-galactanases from bacteria and fungi. The homologous genes are conserved in several strains of B. longum subsp. longum and B. longum subsp. infantis but not in other bifidobacteria. Transcriptional analysis revealed that BLLJ_1840 is intensively induced with BLLJ_1841, an endo-β-1,6-galactanase candidate, in the presence of LWAG. This is the first report of exo-β-1,3-galactanase in bifidobacteria, which is an enzyme used for the acquisition of AG-II in B. longum subsp. longum.

Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-l-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-l-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-l-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-l-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn(2+) coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-l-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

Preparation of p-nitrophenyl β-l-arabinofuranoside as a substrate of β-l-arabinofuranosidase

Synthesis of p-nitrophenyl β-l-arabinofuranoside 1 as the substrate for novel β-l-arabinofuranosidase has been achieved by using both our inter- and intra-molecular glycosylation methodologies. Although the intermolecular glycosylation with l-Araf donors 3 and 4 resulted in a mixture of both α- and β-isomers, NAP ether-mediated IAD with 3 and 6 afforded the desired β-l-arabinofuranoside stereospecifically which was confirmed by NMR analysis on the (3)JH1-H2 coupling constant and (13)C chemical shift of C1. As expected, 1 has been revealed to be an efficient substrate in the biological study of a novel β-arabinofuranosidase such as HypBA1 with higher apparent affinity compared with other reported substrates.

Two Novel α-L-Arabinofuranosidases from Bifidobacterium longum subsp. longum belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan

Arabinose-containing poly-or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum, though their degradation pathways are poorly understood. In this study, we found that the gene expression levels of bllj 1852 and bllj 1853 from B. longum subsp. longum JCM 1217 were enhanced in the presence of arabinan. Both genes encode previously uncharacterized glycoside hydrolase (GH) family 43 enzymes. Subsequently, we cloned those genes and characterized the recombinant enzymes expressed in Escherichia coli. Both enzymes exhibited α-L-arabinofuranosidase activity toward synthetic p-nitrophenyl glycoside, but the specificities for L-arabinofuranosyl linkages were different. BLLJ_1852 catalyzed the hydrolysis of α1,2- and α1,3-L-arabinofuranosyl linkages found in the side chains of arabinan and arabinoxylan. BLLJ_1852 released L-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. BLLJ_1853 catalyzed the hydrolysis of α1,5-L-arabinofuranosyl linkages found on the arabinan backbone. BLLJ_1853 released L-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Both enzyme activities were largely suppressed with EDTA treatment, suggesting that they require divalent metal ions. BLLJ_1852 was moderately activated in the presence of all divalent cations tested, whereas BLLJ_1853 activity was inhibited by Cu2+. The GH43 domains of BLLJ_1852 and BLLJ_1853 are classified into GH43 subfamilies 27 and 22, respectively, but hardly share similarity with other biochemically characterized members in the corresponding subfamilies. IMPORTANCE We identified two novel α-L-arabinofuranosidases from B. longum subsp. longum JCM 1217 that act on different linkages in arabinan. These enzymes may be required for efficient degradation and assimilation of arabinan in the probiotic bifidobacteria. The genes encoding these enzymes are located side-by-side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.