Background:Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes. Results: AgaSK is a protein coupling galactosidase and sucrose kinase activity. The structure of the galactosidase domain sheds light onto substrate recognition. Conclusion: AgaSK produces sucrose-6-phosphate directly from raffinose. Significance: Production of sucrose-6-phosphate directly from raffinose points toward a novel glycolytic pathway in bacteria.

dRuminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an ␣-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14 -21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for ␣-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the ␣-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with ␣-(1,6) regioselectivity, leading to the formation of product structures up to [Hex] 12 and [Hex] 8 , respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.T he human gut is colonized by a complex, diverse, and dynamic community of microbes that continuously interact with the host (30). The majority belongs to only four bacterial divisions, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, whereas other minor taxonomic divisions are quite diverse (19,43,62). Several ecological studies have shown that microbial symbionts have adapted to maximize metabolic access to a wide variety of dietary and host-derived carbohydrates (glycans), and competition for these nutrients is considered a major factor shaping the structure-function of the microbiota (33). Recently, a metagenomic analysis of gut microbial communities in humans proposed three predominant variants, or "enterotypes," dominated by Bacteroides, Prevotella, and Ruminococcus (3). A controlledfeeding study showed that enterotype partitioning associates with long-term diets (61). Furthermore, the ability to selectively use prebiotics carbohydrates, ranging from oligosaccharides to polysaccharides, provides a competitive advantage over other bacteria in this ecosystem (28). These studies highlight the importance of understanding precisely how nutrient metabolism serves to maintain a symbiotic relationship between gut bacteria and the host. The genomes of gut bacteria encode a wide array of carbohydrateactive enzymes (CAZymes) that d...

The human gut microbiome plays an essential role in maintaining human health including in degradation of dietary fibres and carbohydrates further used as nutrients by both the host and the gut bacteria. Previously, we identified a polysaccharide utilization loci (PUL) involved in sucrose and raffinose family oligosaccharide (RFO) metabolism from one of the most common Firmicutes present in individuals, Ruminococcus gnavus E1. One of the enzymes encoded by this PUL was annotated as a putative sucrose phosphate phosphorylase ( Rg SPP). In the present study, we have in-depth characterized the heterologously expressed Rg SPP as sucrose 6 F -phosphate phosphorylase (SPP), expanding our knowledge of the glycoside hydrolase GH13_18 subfamily. Specifically, the enzymatic characterization showed a selective activity on sucrose 6 F -phosphate (S6 F P) acting both in phosphorolysis releasing alpha- d -glucose-1-phosphate (G1P) and alpha- d -fructose-6-phosphate (F6P), and in reverse phosphorolysis from G1P and F6P to S6 F P. Interestingly, such a SPP activity had never been observed in gut bacteria before. In addition, a phylogenetic and synteny analysis showed a clustering and a strictly conserved PUL organization specific to gut bacteria. However, a wide prevalence and abundance study with a human metagenomic library showed a correlation between SPP activity and the geographical origin of the individuals and, thus, most likely linked to diet. Rgspp gene overexpression has been observed in mice fed with a high-fat diet suggesting, as observed for humans, that intestine lipid and carbohydrate microbial metabolisms are intertwined. Finally, based on the genomic environment analysis, in vitro and in vivo studies, results provide new insights into the gut microbiota catabolism of sucrose, RFOs and S6 F P.

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 Rg AgaSK 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 Rg AgaSK for RFO’s short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the Rg AgaSK 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 Rg AgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.