Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

Abstract

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...

Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent

“…1. Both strains possess similar number of GH13 enzymes while the E1 strain has a higher number of GH1, GH2 and GH3, thus, together with a higher number of GH36 (α-galactosidase), GH78 (rhamnosidase), GH43 (xylosidase/arabinosidase), GH29 and GH95 (α-fucosidases), and strain-specific GH63 (α-glucosidase), GH16 (β-glucanase), GH91 (inulin fructotransferase), the E1 strain seems to be more adapted to the degradation of a diversified array of dietary carbohydrate-based substrates [42]. In contrast, the R. gnavus ATCC 29149 genome encodes less GHs than E1 but with a higher proportion of enzymes putatively implicated in degradation of host-derived oligosaccharides, including predicted GH33 sialidase and GH98 endo-β-galactosidase, which are absent in the R. gnavus E1 genome, and both predicted to be extracellular.…”

Evaluating the Contribution of Gut Microbiota to the Variation of Porcine Fatness with the Cecum and Fecal Samples

“…R. gnavus plays a pivotal role in UDCA formation in the colon, which regarded as a supplement of the bile acid (Lee et al, 2013). A recent study found that the α-galactosidase 1 (Aga1) and α-galactosidase 2 (Aga2) which are two kinds of the glycoside hydrolase (GH) family from R. gnavus played an indispensable role in the degradation of dietary oligosaccharides and exerted a tremendous fascination on designing of galacto-oligosaccharide (GOS) prebiotics (Cervera-Tison et al, 2012). The studies in human and rat found that R. gnavus was enriched in the obese rats and humans (Petriz et al, 2014; Andoh et al, 2016).…”

Fecal microbiota in pouchitis and ulcerative colitis

“…R. gnavus produces the bacteriocin ruminococcin A, which inhibits the growth of phylogenetically-related species and various bifidobacterial and clostridial species[32]. Ruminococcin A also degrades intestinal mucin[33] and induced α-galactosidase and β-glucuronidase activity in vitro [34]. β-glucuronidase activity generates toxic metabolites in the colon, which provoke local inflammation.…”