1. Volume 25, Issue 9, Pages 1348-1359.e3 (September 2017)
Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of β- 1,3-Glucan Quaternary Structure 
Benjamin Pluvinage, Alexander Fillo, Patricia Massel, Alisdair B. Boraston 
Structure 
Volume 25, Issue 9, Pages e3 (September 2017)
DOI: /j.str
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2. Structure 2017 25, 1348-1359.e3DOI: (10.1016/j.str.2017.06.019)
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3. Figure 1
Activity of the BhGH81 Catalytic Module from Bacillus halodurans BH0236
(A–C) FACE analysis of BhGH81 activity on laminarin, curdlan, pachyman, and scleroglucan. The blank in each gel represents a sample lacking both sugar and enzyme and therefore shows the migration of the 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) label. O/N, overnight.
(D and E) FACE analysis of β-1,3-glucooligosaccharide hydrolysis by BhGH81. The substrates are labeled above the gels while the presence or absence of enzyme in the reaction is indicated below the gels.
In all panels, L2 to L6 indicate standards ranging from laminaribiose up to laminarihexaose. G indicates the glucose standard. The asterisks indicate bands resulting from the ANTS label.
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4. Figure 2 A Product Analysis of Laminarin and Curdlan Hydrolysis
Time course of laminarin (A) and curdlan (B) digestion analyzed by FACE. The time points and presence or absence of enzyme in the reaction are indicated below the gels. In all panels, L2 to L6 indicate standards ranging from laminaribiose up to laminarihexaose. G indicates the glucose standard. The asterisks indicate bands resulting from the ANTS label. See also Figure S1.
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5. Figure 3 Structure of BhGH81 Shown from Two Angles
The N-terminal segment is colored orange and labeled N. The remaining composite domains I–IV are labeled and colored yellow, blue, purple, and green, respectively. In the right panel the active site is shown as a gray surface with putative catalytic residues colored orange and labeled.
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6. Figure 4
Structure of BhGH81 in Complex with Reaction Products from L6 Cleavage
(A) Electron density for oligosaccharides bound to BhGH81 in the L6 product complex. The blue mesh shows the electron density for these sugars as a σa-weighted Fo-Fc omit map contoured at 3σ. The L3 product is shown as blue sticks and the L2 product as orange sticks. Active-site subsites are indicated in red.
(B) The active site in complex with products resulting from L6 cleavage. L3 is shown as blue sticks and L2 as orange sticks. Relevant amino acid side chains are shown as gray sticks, waters as red spheres, and putative hydrogen bonds as gray dashes. The side chain proposed to act as the acid, D466, is colored magenta and the proposed base, E542, is colored blue. Active-site subsites are labeled in red.
(C) A close-up of the catalytic machinery and glucose residue occupying the −1 subsite, which shows its distortion to a 5S1 conformation. The blue mesh shows the electron density for this glucose residue as a σa-weighted Fo-Fc omit map contoured at 3σ.
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7. Figure 5
Structure of BhGH81 in Complex with Reaction Products from Laminarin Cleavage
(A) The blue mesh shows the electron density for the glucose residues as a σa-weighted Fo-Fc omit map contoured at 3σ. The oligosaccharide bound in the ancillary site is shown as green sticks and the oligosaccharides bound in the active site as orange and blue sticks. Active-site subsites are indicated in red numbers and ancillary binding subsites in red uppercase letters.
(B) Two views of the product complex of BhGH81 obtained by soaking crystals in laminarin focusing on the L10 molecule occupying the ancillary binding site near the catalytic site. The additional L3 and L2 products of hydrolysis are shown as transparent blue and orange sticks, respectively (see also Figure S2). The ancillary binding subsites are labeled as red uppercase letters.
(C) A close-up of W615 in the laminarin complex showing how it is sandwiched between glucose residues in the +1 and +2 catalytic sites and glucose residues in the L10 molecule bound in the ancillary binding site. The ancillary binding subsites are labeled as red uppercase letters.
(D) The solvent-exposed surface of BhGH81 in complex with the products of laminarin hydrolysis and colored by electrostatic potential. The L10 molecule is shown as green sticks, the L3 as blue sticks, and the L2 as orange sticks.
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8. Figure 6 Structure of BhGH81E542Q in Complex with L6
(A) The blue mesh shows the electron density for this glucose residue as a σa-weighted Fo-Fc omit map contoured at 3σ. The oligosaccharide bound in the ancillary site is shown as green sticks and the oligosaccharide bound in the active site as orange sticks. Active-site subsites are indicated in red, and ancillary binding subsites in red uppercase letters.
(B) The active site in complex with two molecules of non-hydrolyzed β-1,3-glucooligosaccharides. L5 was modeled into the ancillary binding site and is shown as green sticks. The L6 in the active site is shown as orange sticks. The W615 side chains separating the ancillary site and active site is shown as gray sticks. The proposed acid catalyst, D466, is colored magenta and the position of the proposed base, E542, is colored blue. Waters bridging the oligosaccharides in the ancillary binding site and active site are shown as red spheres, and putative hydrogen bonds as gray dashes. The ancillary binding subsites are labeled as red uppercase letters.
(C) A close-up of the catalytic machinery and glucose residue occupying the −1 subsite, which shows its distortion to a 2,5B conformation. The blue mesh shows the electron density for this glucose residue as a σa-weighted Fo-Fc omit map contoured at 3σ. The catalytic residues are shown as sticks and the putative catalytic water is shown as a red sphere.
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9. Figure 7
Schematic Representing Occupation of the Active-Site Subsites and the Ancillary Binding Sites in the BhGH81E542Q Mutant by β-1,3-Glucooligosaccharides
The normal point of glycosidic bond cleavage is shown by the orange arrows. The red circles indicate glucose residues in the 2,5B conformation of the Michaelis-like complex while the blue circles indicate the 5S1 glucose conformation observed in the product complexes. NR and R refer to the non-reducing and reducing ends of the sugars, respectively. The sugar marked “a” was only partially occupied. +3∗ indicates an ordered glucose residue that does not occupy a clearly defined subsite. N/A indicates no occupation of these binding sites was observed.
See also Figures S3–S6.
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10. Figure 8
Comparison of the Curdlan Triple-Helical Structure with the Surface Properties of BhGH81
(A) The solvent accessible surface of BhGH81 (gray) showing the bound L10 (green), L2 (purple), and L3 (purple) molecules from the laminarin product complex. The triple-helical structural determined for curdlan II is shown as orange, yellow, and blue β-1,3-glucan chains (Chuah et al., 1983). The curdlan triple helix was placed by overlapping the yellow β-1,3-glucan chain in the curdlan structure with the L10 molecule in the BhGH81 complex. The pitch of the helix formed by a single glucan chain in curdlan is ∼16 Å while the separation between adjacent chains in the triple helix is ∼5.3 Å.
(B) A close-up of the BhGH81active site showing that the oligosaccharides engaging the enzyme's catalytic machinery are displaced down into the active site ∼6–7 Å relative to the chains in the modeled curdlan triple helix.
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