α-Galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities

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.

FIGURE 1.

Structure of the α-galactosidase domain of AgaSK, tetrameric assembly, substrate-binding site, and model for full-length AgaSK. A, graphic presentation of the α-galactosidase domain of AgaSK, with the N-terminal domain in deep blue, the catalytic (β/α)₈ barrel in light blue, and the C-terminal in marine blue. Galactose is shown as red sticks. B, tetrameric assembly of AgaSK-tru, with one monomer oriented and colored as in A and the other monomers shown in pink, green, and yellow hues, respectively. C, the α-galactosidase active site. Residues interacting with the product are shown as sticks, with carbons color-coded as in B. Galactose is shown as sticks with green carbons, and the catalytic residues Asp-478 and Asp-540 are shown as sticks with yellow carbons. Oxygen and nitrogen atoms are colored in red and blue, respectively. F_o − F_c electron density calculated prior to incorporation of galactose into the model and contoured at 4 σ is shown in green. D, the association of three monomers within the tetrameric assembly creates a deep tunnel for substrate binding. The substrate-binding groove is shown in surface representation, with different monomers color-coded as in B. Galactose is shown as sticks with yellow carbons, and a model of raffinose, derived from a superposition of AgaSK-tru with S. cerevisiae α-galactosidase 1, Mel1, complexed with raffinose (PDB 3lrm), is shown as sticks with green carbons. E, model for full-length AgaSK. The tetrameric assembly is color-coded as in B. The modeled C-terminal domains are colored in the same hue as the N-terminal domains of each monomer. The polypeptide chain at the junction of both domains has to bend backwards toward the (β/α)₈ barrel of an adjacent subunit to avoid steric clashes. This places each nucleotide-binding domain in proximity to an α-galactosidase active site. Galactose and modeled ATP molecules are shown as red sticks. Two junctions between the galactose and the kinase domain are indicated with red arrows.

FIGURE 2.

Characterization of sugar phosphorylation by AgaSK. A and B, phosphorylation of several sugars was tested in reaction mixtures containing AgaSK, ATP, MgCl₂, [γ-³³P]ATP (0.5 μCi), and different sugars. The radioactive spots were detected on the autoradiogram (A). The sugar spots were detected with 0.1% orcinol (B). Lanes 1, without AgaSK; lanes 2, with AgaSK; lanes 3, with raffinose; lanes 4, with melibiose; lanes 5, with sucrose; lanes 6, with glucose; lanes 7, with fructose; lanes 8, with galactose. C, phosphorylation of raffinose and sucrose was tested in reaction mixtures containing AgaSK, ATP, and MgCl₂. Lane 1, without AgaSK; lane 2, with AgaSK; lane 3, with raffinose; lane 4, with raffinose and 10 μm Gal-DNJ; lane 5, with raffinose and 100 μm Gal-DNJ; lane 6, with sucrose; lane 7, with sucrose and 10 μm Gal-DNJ; lane 8, with sucrose and 100 μm Gal-DNJ. The sugar spots were detected with 0.1% orcinol.

FIGURE 3.

Positive ESI-Q-TOF-MS mass spectra and chemical structure of phosphorylated sugar. All reaction products are deduced from the MS/MS data at m/z 365.107 (sucrose monosodiated form), 445.072 (phosphorylated sucrose monosodiated form), 467.060 (phosphorylated sucrose disodiated form), and 203.018 (galactose monosodiated form). The structural features of phosphorylated sucrose are deduced from an ion m/z 445.059 MS/MS spectrum. A, full scan MS of the sample from enzymatic reaction (solvent ions are indicated by black triangles). B, MS/MS of the sodiated ion at 445,1 with corresponding annotated fragments. In two areas of the MS/MS spectrum, the peak intensity was scaled up 24 and 36 times, respectively. C, chemical structure of the phosphorylated oligosaccharide annotated with corresponding fragments.

FIGURE 4.

Sucrose and raffinose pathways in R. gnavus E1 (adapted from Reid and Abratt (13)). Two pathways for sucrose transport are well characterized, the PTS-dependent sucrose system (left) and the non-PTS transport system (middle). The raffinose pathway (right) could be another possibility to provide sucrose to the bacterium.