Abstract
Plant α-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by α-galactosidases produced by the human gut microbiome. In this environment, the Ruminococcus gnavus E1 symbiont–well-known for various benefit–is able to produce an original RgAgaSK bifunctional enzyme. This enzyme contains an hydrolytic α-galactosidase domain linked to an ATP dependent extra-domain, specifically involved in the α-galactoside hydrolysis and the phosphorylation of the glucose, respectively. However, the multi-modular relationships between both catalytic domains remained hitherto unexplored and has been, consequently, herein investigated. Biochemical characterization of heterologously expressed enzymes either in full-form or in separated domains revealed similar kinetic parameters. These results were supported by molecular modeling studies performed on the whole enzyme in complex with different RFOs. Further enzymatic analysis associated with kinetic degradation of various substrates followed by high pressure anionic exchange chromatography revealed that catalytic efficiency decreased as the number of D-galactosyl moieties branched onto the oligosaccharide increased, suggesting a preference of RgAgaSK for RFO’s short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the RgAgaSK encoding gene whatever the health status of the individuals. Finally, phylogeny and synteny studies suggested a limited spread by horizontal transfer of the clusters’ containing RgAgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.
Highlights
Raffinose family oligosaccharides (RFOs) are mainly found in the seeds of vegetables
To investigate the interactions between both domains of the RgAgaSK enzyme, we generated two variants consisting of each domain individually expressed in Escherichia coli BL21 cells, the N-terminal domain corresponding to the α-galactosidase domain (RgAga, from Ala-1 to Lys-720) and the C-terminal domain corresponding to the kinase domain (RgSK, from Arg-724 to Gln935), respectively
Analysis of the physico-chemical properties of the α-galactosidase domain revealed no difference with the whole protein, whereas according to the kinetic parameters, RgAga appears slightly more efficient for pNPGal hydrolysis compared to RgAgaSK
Summary
Raffinose family oligosaccharides (RFOs) are mainly found in the seeds of vegetables. Dietary RFOs pass through the stomach and the small intestine without major changes to reach the colon almost unchanged before being fermented or degraded by anaerobic commensal bacteria (Martínez-Villaluenga et al, 2008). RFOs were considered to be antinutritional factors, as their consumption in large quantities leaded to negative effects such as abdominal pains, flatulence, bloating and diarrhea, mainly due to their fermentation in the colon (Martínez-Villaluenga et al, 2008). The vision of the RFOs is changing thanks to the in-depth studies carried out in the last decade and they are considered as prebiotics (Collins and Reid, 2016) as their consumption at the right dose could promote the growth of beneficial bacteria including Bifidobacteria and Lactobacilli (Ejby et al, 2016; Collins et al, 2017; Zartl et al, 2018). In the highly competitive ecosystem constituted by the gastrointestinal tract, RFOs represent a selective advantage for organisms capable of metabolizing them to the detriment of others, and for the host health
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