Figures and data
Structural description of GAGase II and GAGase VII.
(A), Overall structures of GAGase II (left) and GAGase VII (right) are shown in carton. The α-helix, β-strand and random coil are colored with yellow, green and gray, respectively. The N-terminal (α/α)7 toroid domain and C-terminal two-layered β-sheet domain were circled and the secondary structure elements are marked nearby. (B), Mn binding site of GAGase II and GAGase VII. The purple sphere presents Mn2+. Mn2+ binding site of GAGase II (left) and GAGase VII (right) is shown in stick. A cut off distance of 3.0 Å was carried out to choose neighboring residues.
Data collection and refinement statistics.
Activity of GAGase II and its mutants.
(A), Activity of site-directed mutants of His and Asp nearby the Mn2+. His and Asp residues nearby the Mn2+ were individually mutated to Ala and the relative activity of each mutant was measured as described in “Materials and methods”. All of the residual activities were evaluated and shown as the relative intensity compared with that of wild type GAGase II. ***: p<0.001. (B), The effects of Mn2+ and chelating reagent of GAGase II were determined using HA (1 mg/ml) as substrate. Error bars represent averages of triplicates ± S.D.
Multiple structural alignments of GAGase II and its structurally similar proteins.
GAGase II (8KHV, blue) was aligned with structurally identified GAGs and alginate lyases, including PL17 family alginate lyase (4NEI, purple), PL39 family alginate lyase (6JP4, olive), PL15 family alginate lyase (3A0O, red), PL12 family heparinase III (4MMH, salmon), PL15 family exoHep (6LJA, gray), PL21 family heparinase II (2FUQ, magenta), PL8 family chondroitin sulfate AC lyase II (1RWA, green), PL23 family chondroitinase (3VSM, yellow) and PL8 family chondroitin sulfate ABC lyase II (2Q1F, pink). The detailed views of the crucial active site residues are shown in stick mode. The root-mean-square deviation (RMSD) between these structures and GAGase II were calculated as 1.342, 1.344, 1.299, 1.242, 1.145, 1.352, 1.235, 1.138 and 1.405 Å based on 171, 148, 141, 111, 99, 87, 60, 22 and 21 pruned atoms, respectively.
Structural homology of GAGase II/GAGase VII with GAG/alginate lyases analysed using DALI.
Catalytic center and substrate binding sites of GAGases.
(A), The crucial catalytic site-directed mutagenesis of GAGase II, GAGase III and GAGase VII. The conserved crucial residues of GAGase II, GAGase III and GAGase VII were individually mutated to Ala. CS-C and alginate were used as substrates for the activity evaluation of GAGase II, GAGase III, GAGase VII and its mutants. The activity of each mutant was detected using gel filtration HPLC on a Superdex Peptide column as described in “Materials and methods”; (B), Molecular docking of GAGase II with a HA hexasaccharide. The molecule docking was carried out with GAGase II and a HA hexasaccharide (PDB code: 1HYA) to predict the substrate binding sites. The binding site residues (green) and hexasaccharide ligand (gray) are showed as sticks. (C), The putative substrate-binding site residues surrounding the docking substrate were individually mutated to Ala and the relative activity of each mutant was shown as the relative intensity compared with that of wild type GAGase II. *: p<0.01, **: p<0.001, ***: p<0.0001, #: the activities of the mutants were too low to be accurately detected and almost completely inactivated.
Catalytic residues comparison of identified GAGs and alginate lyases shared similar structure with GAGase II and GAGase VII
Analysis of a key residue for the alginate-degrading activity of GAGase III
(A), Multiple structural alignment of GAGase II, III and VII. GAGase II (8KHV, green) and GAGase VII (8KHW, skyblue) were aligned with GAGase III (model, gray). The detailed views of crucial catalytic residues are shown in stick mode; (B), Conversed catalytic residues of alginate lyases from PL17 (4NEI, purple), PL39 (6JP4, yellow), PL38 (8BDQ, slate), PL5 (7FHU, cyan) and PL15 (3A0O, pink) family. Residues are shown in stick; (C), Activity assay of GAGase III-H188N and GAGase III-H188A toward CS-C and alginate. The crucial site His188 was mutated to alanine and asparagine, respectively. The activity of each mutant against CS-C and alginate was assessed using gel filtration HPLC on a Superdex Peptide column, as described under “Materials and methods”; (D), Relative activity of GAGase III and its mutants. Three types of GAGs (HA, CS-C and HS) were individually treated with GAGase III and its mutants (GAGase III-H188N and GAGase III-H188A) at 40 °C for 1 h; relative activities of enzymes were determined by detecting the absorbance at 232 nm. Data are shown as the percentage of the activity relative to the wild-type GAGase III. *, p<0.5; **, p<0.01; ***, p<0.001.
Molecular docking of GAGase II with hexasaccharide ligands.
Molecular docking of GAGase II with CS ligands. The molecule docking was carried out with GAGase II and CS ligands, including nonsulfated (PDB code: 2KQO) (left) and 4-O-trisulfated (PDB code: 1C4S) (right) CS hexasaccharide, to investigate the substrate selectivity. The catalytic triplet residues (green) and CS ligand (yellow) are showed as sticks.
Proposed catalytic mechanism of GAGase II and GAGase III.
Take the HA degradation by GAGase II and the polyM degradation by GAGase III for example. Briefly, the substrate is firstly binding to the negatively charged residues near the catalytic sites, the carboxylate group is neutralized by Asn192, Asn191 in GAGase II and His188, Asn189 in GAGase III; His398 in GAGase II and His401 in GAGase III are proposed to work as general base to abstract a proton from the C5 position of GlcUA at +1 position, and Tyr246 in GAGase II and Tyr243 in GAGase III work as general acid to donate the leaving group at −1 position a proton. The arrows indicate the direction of electron transfer, and the C5-C6 double bond on the middle panel indicate the enolate anion intermediate created by proton abstraction at C-5 position.
