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

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-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 α-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 α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(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.

Fig 1

Transcriptional organization of the aga2 operon in R. gnavus E1. (A) Schematic diagram of the aga2 cluster. sucR (RUGNEv3_61261) is annotated as a CcpA-like regulator, scrA1 (RUGNEv3_61260) as a sucrose-specific Glc-like PTS EIIABC, malE1 (RUGNEv3_61259) as a GH13 α-amylase/trehalose_treC, aga2 (RUGNEv3_61258) as a GH36 α-galactosidase, scrA2 (RUGNEv3_61257) as a sucrose-specific PTS EIIBC, and sacA (RUGNEv3_61256) as a GH32 sucrose-6-phophate hydrolase. The general promoter of the aga2 cluster, as determined by RT-PCR, is marked by an arrow. Circles above thick vertical lines indicate potential stem-loop structures that might act as rho-independent transcriptional terminators. The free energy of the thermodynamic ensemble is given on top, expressed as kcal · mol⁻¹. The inset shows the DNA sequence of the catabolite repression-responding promoter located upstream of PTS1. The putative −35 and −10 regions and ribosome-binding site (RBS) are underlined. The catabolite-responsive element (CRE) box is indicated with a black box. (B) Agarose gel electrophoresis of PCR products obtained following RT-PCR of total RNA extracted from gnobiotic rats associated with R. gnavus E1 was performed using primers spanning the intergenic regions of the aga2 cluster (RT⁺). In addition, one pair of primers was designed to amplify an internal region of the gene encoding Aga2, as a positive control. The negative controls (RT⁻) were without reverse transcriptase; the absence of PCR fragments confirmed that the products were amplified from RNA and not from potential contamination by genomic DNA. Lanes 1, 2, 3, 4, 5, and 6 correspond to the sucR-scrA1, scrA1-malE1, malE1-aga2, aga2-scrA2, and scrA2-sacA intergenic regions and an internal region of aga2, respectively. The positions of the primers are shown in panel A. M, DNA ladder size marker (with increments indicated in base pairs).

Fig 2

Unrooted phylogenetic tree of GH36 α-galactosidases from Firmicutes. R. gnavus E1 α-galactosidases Aga1, Aga2, and AgaSK are underlined. Percent bootstrap values are indicated. str, strain.

Fig 3

Homology models of R. gnavus E1 Aga1 and Aga2. (A) Cartoon representation of the monomeric structure models of Aga1 and Aga2, with the N-terminal domains in pink, the catalytic (β/α)₈ barrels in green, and the C-terminal domains in slate blue. Gal and catalytic residues are shown as orange and red sticks, respectively. (B) Cartoon representation of the tetrameric structure models. One monomer is oriented and colored as described for panel A, and the other monomers are colored in yellow, orange, and gray. Galactose and catalytic residues are shown as orange and red sticks, respectively.

Fig 4

Effect of pH (A) and temperature (B) on the α-galactosidase-specific activity (SA) of recombinant R. gnavus E1 Aga1 (dashed lines and rhombuses) and Aga2 (solid lines and triangles). The data represent the means ± standard deviations of the results of at least three independent assays.

Fig 5

Substrate specificities of R. gnavus E1 Aga1 and Aga2. (A) Hydrolysis of pNP-pyranosides by R. gnavus E1 Aga1 (light gray) and Aga2 (dark gray). The recombinant enzymes were incubated with different sugars linked to pNP, and the cleavage of the linkage was estimated as the amount of pNP released in the reaction mixture. (B) Hydrolysis of natural substrates by R. gnavus E1 Aga1 (light gray) and Aga2 (dark gray). The specific activity was determined by quantifying the amounts of Gal released during incubation with the recombinant enzymes. (C) HPAEC-PAD analysis of hydrolysis products of Raf (12 mM) incubated for 4 h with Aga1 (114 nM; panel Ci) or Aga2 (234 nM; panel Cii) or a no-enzyme control (panel Ciii). All samples were diluted 1/10 prior to injection. Panel Civ shows an overlay of 0.5 mM standards of Raf (solid line), Suc (dashed line), and Gal (dotted line). nC, nanocoloumbs.

Fig 6

Close-up view of R. gnavus E1 Aga1 (A) and Aga2 (B) active sites. Raffinose, derived from a superposition of the models with S. cerevisiae_α-galactosidase 1, Mel1, complexed with Raf (PDB accession no. 3lrm), is shown as sticks with gray carbons and red oxygens. Residues interacting with the substrate are shown as blue sticks, and residues originating from a neighboring subunit are depicted as yellow sticks. Catalytic residues are shown in red. Hydrogen bonding interactions with a distance threshold of 3.25 Å are shown as dashed lines, and substrate binding subsites are indicated in bold.

Fig 7

HPAEC-PAD analysis of self-condensation products of melibiose (2 M) incubated with Aga1 (15.7 nM). (A) Standards Glc, Gal, and Mel at 500 μM; (B) no-enzyme control after 150 h; (C) reaction after 1 h; (D) reaction after 10 h; (E) reaction after 24 h; (F) reaction after 150 h.