A Bacteroidetes locus dedicated to fungal 1,6-β-glucan degradation: Unique substrate conformation drives specificity of the key endo-1,6-β-glucanase

Abstract

Glycans are major nutrients available to the human gut microbiota. The Bacteroides are generalist glycan degraders, and this function is mediated largely by polysaccharide utilization loci (PULs). The genomes of several Bacteroides species contain a PUL, PUL_{1,6-β-glucan}, that was predicted to target mixed linked plant 1,3;1,4-β-glucans. To test this hypothesis we characterized the proteins encoded by this locus in Bacteroides thetaiotaomicron, a member of the human gut microbiota. We show here that PUL_{1,6-β-glucan} does not orchestrate the degradation of a plant polysaccharide but targets a fungal cell wall glycan, 1,6-β-glucan, which is a growth substrate for the bacterium. The locus is up-regulated by 1,6-β-glucan and encodes two enzymes, a surface endo-1,6-β-glucanase, BT3312, and a periplasmic β-glucosidase that targets primarily 1,6-β-glucans. The non-catalytic proteins encoded by PUL_{1,6-β-glucan} target 1,6-β-glucans and comprise a surface glycan-binding protein and a SusD homologue that delivers glycans to the outer membrane transporter. We identified the central role of the endo-1,6-β-glucanase in 1,6-β-glucan depolymerization by deleting bt3312, which prevented the growth of B. thetaiotaomicron on 1,6-β-glucan. The crystal structure of BT3312 in complex with β-glucosyl-1,6-deoxynojirimycin revealed a TIM barrel catalytic domain that contains a deep substrate-binding cleft tailored to accommodate the hook-like structure adopted by 1,6-β-glucan. Specificity is driven by the complementarity of the enzyme active site cleft and the conformation of the substrate. We also noted that PUL_{1,6-β-glucan} is syntenic to many PULs from other Bacteroidetes, suggesting that utilization of yeast and fungal cell wall 1,6-β-glucans is a widespread adaptation within the human microbiota.

Keywords: X-ray crystallography; carbohydrate-binding protein; glycosidase; glycoside hydrolase; microbiome.

Figure 1.

The 1,6-β-glucan polysaccharide utilization loci from B. thetaiotaomicron and B. ovatus. A, schematic of the 1,6-β-glucan polysaccharide utilization loci (PUL_{1,6-β-glucan}). The percentage identity at protein level between the PULs is indicated. B and C, RT-PCR of PUL_{1,6-β-glucan} genes in B. thetaiotaomicron (B) and B. ovatus (C) grown on 5 mg ml⁻¹ glucose or 1,6-β-glucan, shown as fold change in expression versus glucose. D, HPAEC-PAD analysis of soluble glycans in the culture supernatant during growth of B. thetaiotaomicron on 1,6-β-glucan as shown in E. The A₆₀₀ at the time of sample is indicated. Oligosaccharides were eluted from the column with a 20% gradient of 100 mm NaOH. OD, optical density. E, growth of B. thetaiotaomicron WT or bt3312 deletion strain (Δbt3312) on minimal medium plus 0.5% (w/v) 1,6-β-glucan, or 1,6-β-glucan digested with 50 nm BT3312 for 5 min and then boiled to inactivate enzymes. F, HPAEC-PAD analysis of whole cell assays of WT or Δbt3312 glucose grown cells (black and red) and WT or Δbt3312 1,6-β-glucan digest grown cells (blue and orange) against 2 mg ml⁻¹ 1,6-β-glucan. Oligosaccharides were eluted from the column with a 60% gradient of 100 mm NaOH.

Figure 2.

HPAEC-PAD analysis of BT3312. BT3312 (50 nm) was incubated with 2 mg ml⁻¹ 1,6-β-glucan at 37 °C as described under “Materials and methods.” Samples were taken at the indicated time points and analyzed by HPAEC-PAD.

Figure 3.

The structure of BT3312 in complex with GlcDNJ. A, cartoon representation of the structure. B, active site residues of BT3312 (green) with GlcDNJ (yellow) and electron density 2mF_o − DF_c map contoured at 1.5 σ (blue). C, 2D representation of the active site with hydrogen bonds between protein and ligand at −2 and −1 subsites depicted. D, surface, colored gray, with ligand in yellow and catalytic residues Glu²³⁶ and Glu³³⁷ in magenta. The U-shaped active site is highlighted with a black dashed line.

Figure 4.

Comparison of BT3312 with other GH30 enzymes. A, surface representations with ligand of BT3312, H. sapiens β-glucosylceramidase (PDB code 2V3D) and the D. chrysanthemi glucuronoxylanase (PDB code 2Y24). B, comparison of active site conservation between BT3312 (green), 2Y24 (pink), and 2V3D (orange).

Figure 5.

Isothermal titration calorimetry of glycan-binding proteins. Shown are representative traces of the binding of 50 μm SusD BT3311 (left panel) or SGBP BT3313 (right panel) to 5 mg ml⁻¹ 1,6-β-glucan in 50 mm HEPES, pH 7.5 (estimated at 1 mm to fit the data).

Figure 6.

Distribution of PUL_{1,6-β-glucan} in Bacteroidetes. PULs with synteny to PUL_{1,6-β-glucan} were identified using the Integrated Microbial Genome and Microbiome sample database at www.img.jgi.doe.gov using the Gene Ortholog Neighborhood viewer and PULDB at www.cazy.org/PULDB.⁵ Example PULs from strains that can and cannot grow on α-mannan are labeled. For those strains that have not been tested for growth on α-mannan, www.cazy.org was searched for genes encoding GH76 α-mannanases and GH38 α-mannosidases; those strains lacking the genes were considered as unlikely to grow on α-mannan are indicated by asterisks. The functions or enzyme families of the gene products are indicated. The basis for assigning genes encoding SGBPs was based on significant sequence identity (>35%) with BT3313 (SGBP of B. thetaiotaomicron) and the presence of a lipoprotein signal peptide. The gray genes encode proteins of unknown function.

Figure 7.

Cartoon representation of 1,6-β-glucan degradation by B. thetaiotaomicron. A schematic of 1,6-β-glucan degradation by B. thetaiotaomicron is shown. Gene products are colored as in Fig. 1A. 1,6-β-Glucan is represented by blue circles. The inner membrane transporter to transport monosaccharides into the cytoplasm for fermentation is assumed to exist but is not within PUL_{1,6-β-glucan}.