Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

Abstract

Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for α-l-arabinopyranoside and describe the first representative of this subfamily. We found that this enzyme (Arap42B) from a probiotic species cannot hydrolyze β-galactosides. However,Arap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to α-l-arabinopyranosyl-(1,6)-β-d-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus is not hydrolyzed by classical GH42 β-galactosidases. X-ray crystallography revealed a unique Trp--Trp sequence motif at the Arap42B active site, as compared with a Phe--His motif in classical GH42 β-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Ara lacking this carbon. Automated docking of paeonolide revealed that it can fit into the Ara42B active site. The Glc moiety of paeonolide stacks onto the aromatic ring of the Trp at subsite +1 and C4-OH is hydrogen bonded with Asp Moreover, the aglycone stacks against Phe from the neighboring monomer in the Ara42B trimer, forming a proposed subsite +2. These results further support the notion that evolution of metabolic specialization can be tracked at the structural level in key enzymes facilitating degradation of specific glycans in an ecological niche.

Structure and evolution of the bifidobacterial carbohydrate metabolism proteins and enzymes

“…in Lactobacillus acidophilus [ 85 ]. Recently, a new GH42 α- l -arabinopyranosidase from B. lactis ( Bl Arap42B) was discovered [ 86 ]. Bl Arap42B releases α- l -arabinopyranoside from bioactive plant glycosides, e.g.…”

Crystal structure of a homotrimeric verrucomicrobial exo-β-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes

“…While the overall structure of the Op5 Man5 homotrimer is similar to that of the GH42 β -galactosidases, the active sites are more diverse. Besides the trimeric β -galactosidases, the only other enzymatic activity found in family GH42 is the α -L-arabinopyranosidase from Bifidobacterium animalis ( Bl Arap42B; PDB code 5XB7 ( Viborg et al, 2017 ), also a homotrimer, and the enzyme displays structural similarity to Op5 Man5 comparable to that of the GH42 β -galactosidases: 2.2 Å for 261 (of 349) aligned Cα positions (22.5% sequence identity) for domain A; and 2.4 Å for 165 (of 204) aligned Cα positions (15.2% sequence identity) for domain B.…”

Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

“…Domain B bears a striking resemblance to the trimerization domain of GH42 enzymes. In GH42 structures (15,18,20) there are a number of interactions between the trimerization domain and the catalytic domain which support trimeric quaternary structure. Likewise, domain B of Bs164 interacts over a surface area of 1380 Å 2 per protomer that includes seven residues involved in direct hydrogen bonds and two salt bridges between chains.…”

“…Unlike the first two domains, the function of the C-terminal β-sandwich domain is much more difficult to infer from the structure. This domain is also present in GH42 enzymes (15,18,20) however, the role of this domain within GH42s is unknown.…”