Abstract
α-Galactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, α-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically phosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.
Highlights
Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes
R. gnavus E1 Is Able to Metabolize Raffinose in the Intestinal Tract—R. gnavus E1 is a Gram-positive bacterium found in the human alimentary tract
The products of agaE, agaF, and agaG could constitute part of an ABC importer specific for raffinose. They present significant homologies with the MsmFII, MsmGII, and MsmEII proteins, the high affinity solute-binding protein, and the transmembrane domains of an ABC transporter involved in raffinose uptake in L. acidophilus NCFM [10]
Summary
An abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes. GH36 members originate from bacteria, fungi, and plants and carry out hydrolysis with a net retention of the anomeric configuration, which is why many enzymes from this family have been shown to possess transglycosylation activity [21,22,23,24,25,26,27] For their ability to hydrolyze ␣-galactosides non-digestible by humans and to synthesize diverse oligosaccharides, GH36 ␣-galactosidases have potential applications for the production of prebiotics. The enzyme AgaSK is composed of a GH36 ␣-galactosidase and a kinase domain and is able to release galactose from ␣-galactosides and to phosphorylate sucrose provided either by raffinose hydrolysis or by the environmental medium. Nisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have