Table 1.
Kinetic parameters of Lin1840r for β-linked gluco-oligosaccharides and pNP-β-Glc.
Table 2.
Inhibition constants for inhibitors.
Fig 1.
Overall structure of Lin1840r.
(A) Lin1840r is a dimer composed of subunits A and B. Domains 1 ((β/α)8 triose phosphate isomerase barrel fold, amino acids 1–339), 2 ((α/β)6 sandwich fold, amino acids 360–543), and 3 (fibronectin type III (FnIII) family, amino acids 605–723) in subunit A are presented in cyan, orange, and magenta, respectively. The loop extending to the active center in subunit B (amino acids 543–604), and the linker region that connects domains 1 and 2 (amino acids 340–359) are shown in red and black, respectively. The corresponding three domains, loop, and linker in subunit B are presented in semitransparent blue, pink, and yellow, brown, and gray, respectively. Mg2+ ions are shown as green spheres. N-terminal and C-terminal regions of both subunits are denoted by N and C, respectively.
Fig 2.
Complexes with GDL (A), and IFG (B).
The two subunits comprising the catalytic pocket are shown in cyan and yellow. Residues involved in ligand binding are shown as sticks. The Leu39–Met47 loop seen in the IFG complex is shown in magenta and the disordered loop region (Gly32–Glu38) are shown in a black dotted line. Inhibitors and glycerol are represented as green and orange sticks, respectively. Fo−Fc electron density maps of GDL and IFG are shown as a gray mesh (contoured at 3.0σ and 4.0σ, respectively). GDL and glycerol (A) and IFG (B) were omitted for calculation of the Fo−Fc maps. Hydrogen bonds are presented as blue dotted lines.
Fig 3.
D270A-Sop2 and D270A-Lam2 complexes.
(A, B) Substrate pockets of D270A-Sop2 (A) and D270A-Lam2 (B) complexes. Ligands are shown as green sticks. Acid-base residues are shown in blue. (Left) Electron density maps of ligands. The Fo−Fc electron density maps of the ligands are presented as a gray mesh (contoured at 3.0σ). Sop2 and Lam2 are omitted for calculation of the Fo−Fc maps. Several hydroxy groups of ligands are labeled. Hydrogen bonds are presented as blue dotted lines. Lam2 in the D270A-Lam2 complex (B) is a mixture of both anomers. Distances between Arg572 and ligands are summarized in S3 Table. (Right) Conformations of ligands at subsite −1. The position of the catalytic nucleophile (Asp270) determined on superpositioning with apo WT is shown in red. The distances between two atoms linked by red dotted lines are given. (C) Comparison of conformation of Sop2 with those of ligands in Michaelis complex and transition state-like complexes at subsite −1. Sop2, Cel2F-DNP in BaCel5A (Michaelis complex), and PheGlcIm in HvExoI (transition state) are shown in green, yellow, and cyan sticks, respectively. Catalytic residues of Lin1840r, HvExoI, and BaCel5A are shown in white, bright orange, and light blue, respectively.
Fig 4.
Comparison of the active centers in Lin1840r and GH3 BGLs.
Apo WT Lin1840r is superimposed on (A) JMB19063, (B) HvExoI (PDB code 1IEX), (C) KmBglI (PDB code 3AC0), (D) AaBGL1 (PDB code 4IIG), (E) TrCel3A (PDB code 3ZYZ), and (F) TnBgl3B (PDB code 2X41). (Left) Domains 1 and 2 of apo WT Lin1840r are shown in cyan, and the loop extending from the other subunit is shown in yellow. The corresponding domains and loop of JMB19063 are shown in pink and green, respectively. HvExoI, KmBglI, AaBGL1, TrCel3A, and TnBgl3B are colored magenta, blue, brown, gray, and orange, respectively. Residues constituting the subsites are represented as sticks. The residue names in Lin1840r and the other enzymes are shown in red and black, respectively. (Right) The D270A-Sop2 complex was used in place of the apo WT enzyme. D270A is shown as a surface representation. The superimposed GH3 BGLs are shown as sticks. Sop2 in the D270A-Sop2 complex is represented as a green stick. All ligands shown in the left figures are omitted in the right figures. (A) An anomeric hydroxy group of Sop2 is labeled.
Fig 5.
Comparison of conformations of ligands in Lin1840r and HvExoI.
Hydrogen bonds are represented as blue dotted lines. Subsites −1, +1, and +2 are labeled. (A) Comparison of Sop2 and Lam2 in the D270A mutant. Arg572 and Sop2 in the D270A-Sop2 complex are shown in cyan and green, respectively. Arg572 and Lam2 of both anomers in the D270A-Lam2 complex are colored orange and blue, respectively. Putative subsite +2 for Sopns and potential subsite +2 for Lamns are shown in a solid and dotted circle, respectively. An anomeric hydroxy group of a reducing-end glucoside in Sop2 is labeled. (B) Comparison of Sop2 in the D270A mutant and thioCel2 in HvExoI. The residue and ligand in the D270A-Sop2 complex are shown in the same as in (A). Arg291 and thioCel2 in HvExoI-thioCel2 are presented in gray and magenta, respectively.