Molecular Cloning and Characterization of a β-l-Arabinobiosidase in Bifidobacterium longum That Belongs to a Novel Glycoside Hydrolase Family

Abstract

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.

Functional Analysis of Degradative Enzymes for Hydroxyproline-linked ^|^beta;-L-Arabinofuranosides in Bifidobacterium longum

“…In various synthetic pNP substrates and natural polysaccharides, the enzyme was able to release disaccharide composed of L-arabinose from commercial arabinan and debranched arabinan (16). However, NMR analysis revealed that the structure of the product was Arafβ1-2Araf (β-Ara 2 ) with β-anomeric configuration, which has never been found in arabinan.…”

Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1)

“…In the hydrolysis of Hyp-linked β-L-arabinooligosaccharides, HypBA2 releases the β-1,2-linked Araf disaccharide (β-Ara 2 ) from Araf-β-1,2-Araf-β1,2-Arafβ-Hyp (Ara 3 -Hyp). HypBA1 subsequently liberates L-arabinoses by hydrolyzing β-Ara 2 [10,11]. In addition, Ara-, Ara 2 -and Ara 3 -Hyp as well as Ara 2 -and Ara-Me (methanol) are also preferred substrates for HypBA1, but Ara 4 -Hyp, extensin, potato lectin, pNP-arabino-, galacto-, glucoand xylo-pyranosides are not.…”

“…In the previous studies, the β-L-arabinofuranosidase (HypBA1, belonging to GH127 family) and β-L-arabinobiosidase (HypBA2, belonging to GH121 family) from Bifidobacterium longum JCM 1217 that participate in the β-L-arabinooligosaccharides metabolisms have been characterized [10,11]. In the hydrolysis of Hyp-linked β-L-arabinooligosaccharides, HypBA2 releases the β-1,2-linked Araf disaccharide (β-Ara 2 ) from Araf-β-1,2-Araf-β1,2-Arafβ-Hyp (Ara 3 -Hyp).…”

Crystallization and preliminary X-ray diffraction analysis of a novel β-L-arabinofuranosidase (HypBA1) fromBifidobacterium longum

“…In the hydrolysis of Hyp-linked -l-arabinooligosaccharides, HypBA2 releases the -1,2-linked Araf disaccharide ( -Ara 2 ) from Araf--1,2-Araf--1,2-Araf -Hyp (Ara 3 -Hyp). Subsequently, HypBA1 liberates the l-arabinoses by hydrolyzing -Ara 2 (Fujita, Sakamoto et al, 2011;Fujita, Takashi et al, 2011). HypBA1 has attracted much attention because no similar crystal structure to HypBA1 has been solved and its catalytic mechanism is still unclear.…”

“…Recently, -l-arabinofuranosidase and -l-arabinobiosidase (HypBA1 and HypBA2), identified from Bifidobacterium longum JCM 1217, have been found to be involved in the metabolic pathway of -l-arabinooligosaccharides (Fujita, Takashi et al, 2011;Fujita, Sakamoto et al, 2011). In the hydrolysis of Hyp-linked -l-arabinooligosaccharides, HypBA2 releases the -1,2-linked Araf disaccharide ( -Ara 2 ) from Araf--1,2-Araf--1,2-Araf -Hyp (Ara 3 -Hyp).…”