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Page 2 of 15 Sasaki et al. Microbiome Res Rep 2023;2:12 https://dx.doi.org/10.20517/mrr.2023.08

Results: MCC10289_0425 was identified to be an unprecedented 3-O-β-L-arabinopyranosyl-α-L-
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arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn to Tyr) at the
catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B.
pseudocatenulatum growth.

Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate
specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant
polysaccharide structures.

Keywords: Bifidobacterium pseudocatenulatum, arabinogalactan protein, type II arabinogalactan, glycoside hydrolase

INTRODUCTION
Bifidobacteria are symbionts in the human gut and produce several carbohydrate-hydrolyzing enzymes to
break down sugars. To control the presence and proliferation of beneficial bacteria in the intestinal tract, it
is essential to understand the degradation and metabolic basis for individual sugar sources, such as milk
oligosaccharides and dietary fibers. Bifidobacteria generally utilize oligosaccharides with relatively low
molecular weight as an energy source and receive degradative products from other symbiont bacteria, such
[1]
as Bacteroides . However, several Bifidobacterium species, particularly the adult type (Bifidobacterium
longum subsp. longum, B. adolescentis, B. pseudocatenulatum, and B. catenulatum), possess extracellular
carbohydrate-hydrolyzing enzymes that can access polysaccharides and glycoproteins directly, thereby
enabling the production of transportable small saccharides from those such as resistant starch , arabinan ,
[3]
[2]
[7-9]
[10]
[5,6]
[4]
mannan , extensin , arabinogalactan protein (AGP) , and arabinoxylan .
The structure of AGP is common in higher plant cell walls. In particular, the structures of type II
arabinogalactan (AG) moieties from gum arabic and larch wood have been well studied and comprise a β-
1,3-galactan backbone and β-1,6-galactan side chains with several modifications of other sugars [11-13] .
Recently, we elucidated the molecular basis of assimilating gum arabic AGP in B. longum JCM7052 . The
[8,9]
extracellular glycoside hydrolase (GH) family 39 enzyme "3-O-α-D-galactosyl-α-L-arabinofuranosidase
(GAfase)" can act on gum arabic AGP and facilitate the action of other enzymes for degrading the AGP
backbone and modified sugar. Gum arabic AGP consists of α-D-Gal-(1→3)-α-L-Araf-(1→3) and β-L-Arap-
(1→3)-α-L-Araf-(1→3) structures, and larch AGP consists of β-L-Arap-(1→3)-α-L-Araf-(1→3) structure in
the side chain. GAfase weakly cleaves β-L-Arap-(1→3)-α-L-Araf-(1→3) linkage in addition to α-D-Gal-(1→
3)-α-L-Araf-(1→3) linkage due to their structural similarity. A homology search revealed that GAfase
homolog genes are found across other Bifidobacterium species. Moreover, based on sequence identity and
peripheral genetic composition, the activities of these homologous enzymes were predicted to be different
from GAfase.

In this study, we performed a functional analysis of B. pseudocatenulatum MCC10289_0425, which has 60%
amino acid sequence identity to GAfase, a novel 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase
(AAfase). AAfase preferentially releases β-L-Arap-(1→3)-L-Ara over α-D-Gal-(1→3)-L-Ara; although its
function overlaps with that of GAfase from B. longum, both enzymes have distinct substrate specificities.
Furthermore, a mutagenesis study revealed the critical amino acid that governs the differentiation of
substrate specificity between AAfase and GAfase. We discussed an example demonstrating diversified
specificity in GH39 within the Bifidobacterium genus. Bifidobacteria might have acquired different fitness
abilities to respond to diverse plant polysaccharide structures.