Three structurally and functionally distinct β-glucuronidases from the human gut microbe Bacteroides uniformis

Abstract

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct β-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.

Keywords: GI tract; bacteroides; carbohydrate metabolism; crystallography; drug reactivation; enzyme structure; glucuronidase; glycoside hydrolase; microbiome; structure-function.

Figure 1.

Discovery and analysis of GUS genes in B. uniformis strain 3978 T3 i. A, schematic for the discovery of GUS enzymes from B. uniformis. B, genetic organization of a PUL from B. uniformis reveals 3 glycoside hydrolases with sequence features indicating GUS function, as well as other glucuronic acid-metabolizing enzymes. C, sequence alignment of EcGUS, BuGUS-1, BuGUS-2, and BuGUS-3 reveals distinct loop classes between these putative GUS enzymes. HTCS, hybrid two-component system; MO, mannonate oxidase; MD, mannonate dehydratase; susC/D, starch utilization system C/D.

Figure 2.

Structural analysis of BuGUS-1, BuGUS-2, and BuGUS-3 reveals distinct tertiary and active site structure. A, tertiary structure of BuGUS-1 with the sugar acid-recognizing NXK motif highlighted as green spheres and catalytic glutamates as deep salmon spheres and zoom-in of active site with unique active site residues highlighted in yellow. B, tertiary structure of BuGUS-2 with core fold highlighted in magenta and additional C-terminal domains in green (DUF1) and yellow (CBM57) and zoom-in of the active site. C, tertiary structure of BuGUS-3 with core fold in blue, the sugar acid-recognizing NXK motif highlighted as green spheres, and catalytic glutamates as deep salmon spheres and additional C-terminal domains in green (DUF1) and yellow (DUF2) and zoom-in of the active site with unique active side residues highlighted in yellow.

Figure 3.

Analysis of quaternary structures and their influence on the active site architecture of EcGUS and BuGUS-1. A, tetramer of EcGUS with zoom-in of the tetramer interface that reveals the hydrophobic pocket around the active site situated at the interface of C-terminal regions. B, tetramer of BuGUS-1 with zoom-in of the tetramer interface revealing a solvent-exposed active site.

Figure 4.

Quaternary structure of BuGUS-2 and BuGUS-3 and structural analysis of C-terminal domains. A, BuGUS-2 dimer with core fold shown in magenta, DUF1 in green, and CBM57 domains in yellow with active site glutamates and the NXK motif shown as deep salmon and green spheres, respectively. B, BuGUS-3 dimer with core fold in blue, DUF1 in green, and DUF2 in yellow with catalytic glutamates and NxK motif in deep salmon and green spheres, respectively. C, CBM57 of BuGUS-2 shown with disordered loop shown as dotted line. D, structure of BuGUS-3 DUF2. E, structural alignment of BuGUS-2 DUF, BuGUS-2 DUF1, and BfGUS DUF.

Figure 5.

Predicted calcium-binding site key for structural and functional integrity of BuGUS-2. A, BuGUS-2 dimer with predicted calcium-binding site (green sphere) 24 Å away from active site glutamates. B, the predicted calcium ion is contacted by Asp-176, Asp-341, Asp-367, and three water molecules. Distances are shown in angstroms. C, active site overlay of WT and calcium-binding mutant of BuGUS-2 reveals conformational changes that preclude functional activity. Distances are shown in angstroms. D, progress curves of BuGUS-2 activity reveal that mutation of the predicted calcium-binding site results in the same loss of function as mutation of essential active site residues.

Figure 6.

Polysaccharide cleavage by BuGUS-1, BuGUS-2, and BuGUS-3. A, percent cleavage for BuGUS-1, BuGUS-2, and BuGUS-3 with an acetylated and sulfated heparin-like substrates for 3 h, pH 6.5. n = 3, ± S.D. NA, no activity. B, schematic structures of the pure synthetic polysaccharides utilized to measure polysaccharide processing by BuGUS enzymes. GlcA, glucuronic acid; GlcNS, N-sulfoglucosamine; GlcNS, N-sulfoglucosamine-6-sulfate; p-PNP, p-nitrophenol.

Figure 7.

Structural analysis of liganded BuGUS-1 and BuGUS-2 reveal chemical complementarity to GlcA. A, PTG bound to BuGUS-1 with mF_o − DF_c simple omit density shown at 2.5 σ with NXK motif shown in green, catalytic glutamates in deep salmon, and Tyr-56 from an adjacent monomer in pale cyan. B, overlay of PTG bound (opaque) and apo (transparent) BuGUS-1 active site reveals significant conformational shifts to catalytic acid/base Glu-421 as well as two nearby residues Lys-454 and Glu-453 to accommodate the large sulfur atom present in PTG. C, BuGUS-1 bound to GlcA with mF_o − DF_c simple omit density shown at 2.5 σ. D, BuGUS-1 active site with GlcA shown in the plane of the ring reveals an α configuration that forms a hydrogen bond with the catalytic nucleophile Glu-508. Distances shown are in units of angstroms.

Figure 8.

Kinetic and structural analysis of SN-38-G hydrolysis reveals the importance of the N-terminal loop in BuGUS-1. A, BuGUS-1 tetramer with adjacent N-terminal loop highlighted in red, catalytic glutamates in deep salmon, and the NXK motif in green. Zoom-in of active site with SN-38-G manually docked in the active site of BuGUS-1 based on the PTG-bound structure. B, catalytic efficiencies k_cat/K_m for EcGUS, BuGUS-1, BuGUS-2, BuGUS-3, BMSP, and BuGUS-1 Δloop with the substrate SN-38-G. C, sequence alignment of BuGUS-1 and BMSP GUS N-terminal loop regions and overlay of BuGUS-1 and BMSP active sites with SN-38-G manually docked. Error bars represent S.D. of n = 3 biological replicates.

Figure 9.

Structural analysis of BuGUS-1 and BuGUS-2 inhibition by d-glucaro-1,4-lactone reveals d-glucaro-1,5-lactone bound instead. A, BuGUS-1 bound to d-glucaro-1,5-lactone with mF_o − DF_c simple omit density shown at 2.5 σ. B, BuGUS-2 bound to d-glucaro-1,5-lactone with mF_o − DF_c simple omit density shown at 1.5 σ. C, proposed mechanisms for the conversion of d-glucaro-1,4-lactone to d-glucaro-1,5-lactone. D, close-up view in the plane of d-glucaro-1,5-lactone reveals planarity at α-carbon and key contact with catalytic acid/base Glu-421. All distances shown are in units of angstroms.