Dataset Information

Characterization of a GH36 α-D-Galactosidase Associated with Assimilation of Gum Arabic in Bifidobacterium longum subsp. longum JCM7052.

ABSTRACT: We recently characterized a 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase) for the release of α-D-Gal-(1→3)-L-Ara from gum arabic arabinogalactan protein (AGP) in Bifidobacterium longum subsp. longum JCM7052. In the present study, we cloned and characterized a neighboring α-galactosidase gene (BLGA_00330; blAga3). It contained an Open Reading Frame of 2151-bp nucleotides encoding 716 amino acids with an estimated molecular mass of 79,587 Da. Recombinant BlAga3 released galactose from α-D-Gal-(1→3)-L-Ara, but not from intact gum arabic AGP, and a little from the related oligosaccharides. The enzyme also showed the activity toward blood group B liner trisaccharide. The specific activity for α-D-Gal-(1→3)-L-Ara was 4.27- and 2.10-fold higher than those for melibiose and raffinose, respectively. The optimal pH and temperature were 6.0 and 50 °C, respectively. BlAga3 is an intracellular α-galactosidase that cleaves α-D-Gal-(1→3)-L-Ara produced by GAfase; it is also responsible for a series of gum arabic AGP degradation in B. longum JCM7052.

SUBMITTER: Sasaki Y

PROVIDER: S-EPMC8367640 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Characterization of a GH36 α-D-Galactosidase Associated with Assimilation of Gum Arabic in Bifidobacterium longum subsp. longum JCM7052.

Sasaki Yuki Y Uchimura Yumi Y Kitahara Kanefumi K Fujita Kiyotaka K

Journal of applied glycoscience 20210611 2

We recently characterized a 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase) for the release of α-D-Gal-(1→3)-L-Ara from gum arabic arabinogalactan protein (AGP) in Bifidobacterium longum subsp. longum JCM7052. In the present study, we cloned and characterized a neighboring α-galactosidase gene (BLGA_00330; blAga3). It contained an Open Reading Frame of 2151-bp nucleotides encoding 716 amino acids with an estimated molecular mass of 79,587 Da. Recombinant BlAga3 re ...[more]

PMID: 34429699

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Project description:Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP and β-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of β-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longumIMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.

Project description:Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and β-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-β-d-GlcpA-(1→6)-β-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-β1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and β1,6-linked α-l-Rhap-(1→4)-β-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-β-d-GlcpA-(1→6)-β-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-β-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Project description:Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited α-l-arabinofuranosidase activity toward p-nitrophenyl-α-l-arabinofuranoside but no activity toward p-nitrophenyl-β-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of α1,2- and α1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the α1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan.IMPORTANCE We identified two novel α-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on α1,2/3-l-arabinofuranosyl linkages, while the latter acts on the α1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. 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.

Dataset Information

Characterization of a GH36 α-D-Galactosidase Associated with Assimilation of Gum Arabic in Bifidobacterium longum subsp. longum JCM7052.

Publications

Characterization of a GH36 α-D-Galactosidase Associated with Assimilation of Gum Arabic in <i>Bifidobacterium longum</i> subsp. <i>longum</i> JCM7052.

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