Crystal structure of glycoside hydrolase family 55 {beta}-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium

Abstract

Glycoside hydrolase family 55 consists of beta-1,3-glucanases mainly from filamentous fungi. A beta-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes beta-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from beta-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two beta-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two beta-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various beta-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two beta-helical domains, demonstrating an unexpected variety of carbohydrate binding modes.

FIGURE 1.

Overall structure of Lam55A. A, ribbon representation of Lam55A monomer in the complex with gluconolactone. B, the view rotated 90° around the horizontal axis from that in A. The N domain, C domain, and linker region are colored blue, green, and magenta, respectively. The disulfide bonds (Cys-5—Cys-424, Cys-73—Cys-77, Cys-539—Cys-549, and Cys-692—Cys-698) are shown in red stick form. The gluconolactone molecule is shown as spheres with carbon and oxygen atoms in white and red, respectively. The loops forming the substrate-binding pocket (T1-4, T1-5, T1-6, and T1-7 from N domain; T3-6, T3-7, T3-8, and T3-9 from C domain) are colored yellow. The T3-5 loop from C domain is colored orange. The zinc atom is shown as an orange sphere. The N-linked sugar chain is shown in cyan stick form. C, the view rotated 90° around the vertical axis from that in A. The torsion angle between the helical axes of the two β-helix domains is indicated.

FIGURE 2.

β-Strands in the two β-helix domains. A, sequence alignment of N and C domains. Arrows for PB1, PB2, and PB3 are colored black, gray, and white, respectively. Identical amino acid residues are boxed in gray. The loops forming the substrate binding pocket and T3-5 loop from C domain are indicated by yellow- and orange-shaded boxes, respectively. Dashed lines indicate regions involved in antiparallel interaction between β-strands of N and C domains. Residues possibly involved in catalysis, Glu-633, Gln-146, Gln-176, and Ser-204, are shown in white type boxed in black. B, superimposition of N domain (blue) and C domains (green). PB1, PB2, and PB3 are colored blue, lime green, and red, respectively. Gluconolactone molecules bound to each domain are shown as spheres. The side chains of residues in N and C domains are shown in blue and orange sticks, respectively. Coils are numbered from the N terminus to the C terminus of each domain. AP indicates a region where β-strands have antiparallel interactions. C, aromatic stack and asparagine ladder found in the N domain.

FIGURE 3.

Binding of gluconolactone to Lam55A. A, stereoview with the F_obs - F_calc omit electron density map contoured at 1.5 σ at the active site. Gluconolactone (white) residues from the N domain (blue) and C domain (green) are shown as stick models. Water molecules are shown as red spheres. Hydrogen bonds are depicted by black dashed lines. Distances between O-1 of gluconolactone and Glu-633 and between C1 and proximal water are indicated by red lines. The main chain carbonyl oxygen of Glu-146 forms a hydrogen bond with the proximal water. B, molecular surface of the gluconolactone complex structure. Amino acid residues highly conserved in biochemically characterized GH55 enzymes, including both exo- and endo-β-1,3-glucanases, are successively colored from yellow (>60%) to orange (>80%) and red (100%). Gluconolactone and aromatic residues located along the groove are shown as stick models. A dashed line indicates a curved groove on the molecular surface, and a circle with green dotted lines indicates the small pocket near the O-6 of gluconolactone.

FIGURE 4.

High performance anion-exchange chromatography analysis of hydrolysis products of laminariheptaose (L7). L7 was incubated with Lam55A for 0 min (A), 1 min (B), 2 min (C), 4 min (D), and 8 min (E) in 100 mm sodium acetate buffer, pH 4.5, at 30 °C, and the reaction mixture was separated as described under “Experimental Procedures.” 2, laminaribiose; 3, laminaritriose; 4, laminaritetraose; 5, laminaripentaose; 6, laminarihexaose; 7, laminariheptaose.

FIGURE 5.

HPLC analysis of hydrolysis products of laminaritriose (L3) and 6-O-glucosyllaminaritriose (LG4). Shown are L3 before (A) and after (B) incubation with Lam55A and LG4 before (C) and after (D) incubation with Lam55A. Each substrate (20 mm) was incubated with Lam55A for 1 min in 100 mm sodium acetate buffer, pH 4.5, at 30 °C, and the reaction mixture was separated as described under “Experimental Procedures.” L2, laminaribiose; Gen, gentiobiose.

FIGURE 6.

Schematic representation of hydrolysis of laminaritriose (A) and 6-O-glucosyl laminaritriose (B) by Lam55A. Lam55A hydrolyzes the glycosidic bond of the glucose residue at the non-reducing end independently of substitution at the O-6 position.

FIGURE 7.

Comparison of active site formation between GH55 Lam55A and PL19 (formerly GH91) BsIFTase. A, superimposition of the structures of Lam55A (blue) and BsIFTase (red and pink for two symmetry-related molecules). B, close-up stereoview of the active site. Selected residues in the active site of Lam55A (Gln-146, Gln-176, Ser-204, and Glu-633) and the catalytic residues of BsIFTase (Asp-233 and Glu-244) are shown as stick models. Gluconolactone bound to Lam55A (carbon atoms in blue) and β-2,1-linked difructosaccharide bound to subsites +1 and +2 of BsIFTase (carbon atoms in pink) are shown as ball-and-stick models. Top views of monomeric Lam55A (C) and homotrimeric BsIFTase (D) are shown. Schematic diagrams are also shown in these panels. The two domains and the linker region of Lam55A and the three symmetry-related chains of BsIFTase are colored differently. The ligands are shown as spheres with the carbon atoms in yellow. The active site is located at the interface between two domains (GH55) or between two adjacent chains (BsIFTase).