Structural and enzymatic characterization of Os3BGlu6, a rice beta-glucosidase hydrolyzing hydrophobic glycosides and (1->3)- and (1->2)-linked disaccharides

Abstract

Glycoside hydrolase family 1 (GH1) beta-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice beta-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-beta-d-fucoside (k(cat)/K(m) = 67 mm(-1) s(-1)), pNP-beta-d-glucoside (k(cat)/K(m) = 6.2 mm(-1) s(-1)), and pNP-beta-d-galactoside (k(cat)/K(m) = 1.6 mm(-1)s(-1)) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-beta-d-glucoside and beta-(1-->3)- and beta-(1-->2)-linked disaccharides and was able to hydrolyze apigenin beta-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-beta-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 A resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended beta-(1-->4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-beta-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.

Figure 1.

Simplified phylogenetic tree of the amino acid sequences of eukaryotic GH1 proteins with known structures and those of rice and Arabidopsis GH1 gene products. The protein sequences of the eukaryotic proteins with known structures are marked with four-character PDB codes for one of their structures, including Trifolium repens cyanogenic β-glucosidase (1CBG; Barrett et al., 1995), Sinapsis alba myrosinase (1MYR; Burmeister et al., 1997), Zea mays ZmGlu1 β-glucosidase (1E1F; Czjzek et al., 2000), Sorghum bicolor Dhr1 dhurrinase (1V02; Verdoucq et al., 2004), Triticum aestivum β-glucosidase (2DGA; Sue et al., 2006), Rauvolfia serpentina strictosidine β-glucosidase (2JF6; Barleben et al., 2007), and Oryza sativa Os3BGlu7 (BGlu1) β-glucosidase (2RGL; Chuenchor et al., 2008) from plants, along with Brevicoryne brassicae myrosinase (1WCG; Husebye et al., 2005), Homo sapiens cytoplasmic (Klotho) β-glucosidase (2E9M; Hayashi et al., 2007), and Phanerochaete chrysosporium (2E3Z; Nijikken et al., 2007), while those encoded in the Arabidopsis and rice genomes are labeled with the systematic names given by Xu et al. (2004) and Opassiri et al. (2006), respectively. One or two example proteins from each plant are given for each of the eight clusters of genes shared by Arabidopsis (At) and rice (Os) and the Arabidopsis-specific clusters At I (β-glucosidases) and At II (myrosinases), with the number of Arabidopsis or rice enzymes in each cluster given in parentheses. These sequences were aligned with all of the Arabidopsis and rice sequences in ClustalX (Thompson et al., 1997), the alignment was manually edited, all but representative sequences were removed, and the tree was calculated by the neighbor-joining method, bootstrapped with 1,000 trials, and then drawn with TreeView (Page, 1996). The grass plastid β-glucosidases, which are not represented in Arabidopsis and rice, are shown in the group marked “Plastid.” Percentage bootstrap reproducibility values are shown on internal branches where they are greater than 60%. Except those marked by asterisks, all external branches represent groups with 100% bootstrap reproducibility. To avoid excess complexity, those groups of sequences marked with asterisks are not monophyletic and represent more branches within the designated cluster than are shown. For a complete phylogenetic analysis of Arabidopsis and rice GH1 proteins, see Opassiri et al. (2006).

Figure 2.

Overall and active site structures of Os3BGlu6. A, Overall ribbon diagram of native Os3BGlu6. The catalytic residues Glu-394 and Glu-178 and one molecule of Tris in two conformations are shown in ball-and-stick representation. The α-helices are colored purple, β-strands are green, and loops are cyan. B, Surface view of the Os3BGlu6 structure showing the active site cleft with the ligand n-octyl-β-d-thioglucopyranoside. The surface is colored by electrostatic potential, with positively charged, negatively charged, and neutral regions colored blue, red, and white respectively. C, Close-up of the electron density of Tris in the active site in stereo view. The Fo-Fc omit electron density map for the Tris is shown as a mesh contoured at I/σ = 3. The side chains of the surrounding amino acids are represented by sticks, and the Tris ligand is represented by balls and sticks. In all frames, oxygen atoms are shown in red, nitrogen in blue, sulfur in dark yellow, protein carbons in yellow, Tris carbons in green or pink, and n-octyl-β-d-thioglucopyranoside carbons in green.

Figure 3.

Binding of 2-fluoroglucoside and n-octyl-β-d-thioglucopyranoside in the Os3BGlu6 active site. A, Stereo view of the superimposition of active site residues at the −1 subsite of the native Os3BGlu6 and Os3BGlu6/G2F complex structures. The residues surrounding the −1 subsite are represented by sticks colored with carbons in blue for native Os3BGlu6 and in yellow for Os3BGlu6/G2F. The G2F moiety bound to the catalytic nucleophile, Glu-394, is represented by balls and sticks with carbon in pink. B, Active site of the Os3BGlu6/G2F complex showing the electron density omit map of G2F and protein-ligand binding interactions. Hydrogen-bonding interactions between G2F and amino acid residues are shown as black dashed lines, while the Fo-Fc omit map of G2F contoured at I/σ = 3.0 is shown as blue mesh. C, Glycone and aglycone interactions of n-octyl-β-d-thioglucopyranoside with Os3BGlu6 residues. Representation of the protein and ligand are as in B, with the Fo-Fc omit map for the ligand contoured at I/σ = 2.5. D, Two views of the superimposition of the G2F and n-octyl-β-d-thioglucopyranoside ligands, showing that the Glc ring is in nearly the same position but is shaped as a relaxed ⁴C₁ chair in G2F (green carbons) and as a ¹S₃ skew boat in n-octyl-β-d-thioglucopyranoside (pink carbons). In all frames, oxygen is in red, nitrogen in blue, fluorine in cyan, and sulfur in yellow.

Figure 4.

Comparison of the Os3BGlu6/n-octyl-β-d-thioglucopyranoside complex active site surface with those of maize ZmGlu1 and rice Os3BGlu7 substrate complexes. A, Active site the Os3BGlu6/n-octyl-β-d-thioglucopyranoside complex structure. The extension of Met-251 to restrict the active site can be seen at lower left. B, The aglycone-binding pocket of the inactive ZmGlu1 E191D mutant/DIMBOAGlc (PDB code 1E56) complex structure. The glucosyl moiety is hidden behind the aglycone. C, Active site of Os3BGlu7 with a docked cellotriose ligand. The smaller Asn-245 (in place of Met-251 in Os3BGlu6) provides a more open cleft for extended oligosaccharide binding. In all frames, active site amino acid residues are shown as sticks behind the gray transparent surface. The ligands are shown in ball-and-stick representations. In the online version, protein carbons are shown in green, n-octyl-β-d-thioglucopyranoside carbons in pink, DIMBOAGlc carbons in blue, cellotriose carbons in yellow, oxygen in red, nitrogen in dark blue, and sulfur in yellow. [See online article for color version of this figure.]