Three crystal structures of endopolygalacturonase I (EndoPG I) from
Stereum purpureum have been determined in this study: the unliganded EndoPG I, the binary and ternary complexes of EndoPG I with galacturonate. Consequently, the structural basis for substrate binding and the catalytic mechanism of EndoPG I have been elucidated by X-ray crystallography. Crystals of deglycosylated EndoPG Ia have been obtained using PEG4000 as precipitate with the hanging-drop vapor diffusion method. The crystal belongs to space group
P1, with unit-cell parameters
a=37.26 Å,
b=46.34 Å ,
c=52.05 Å, α=67.17°, β=72.44°, γ=68.90°. The crystal diffracts to ultra-high (0.96 Å) resolution using synchrotron radiation of SPring-8. Crystal structures of EndoPG I were determined by the multiple wavelength anomalous dispersion (MAD) method. For MAD phasing, three wavelength data sets of K
2PtCl
4 derivative crystal were collected at SPring-8. The structure model was refined anisotropically with SHELXL-97, with an
R factor of 11.4% and an
Rfree factor of 14.0% at 0.96 Å resolution. The enzyme folds into a right-handed parallel β-helix with 10 complete turns. The crystal structures of its binary complex with one
D-galacturonate and its ternary complex with two
D-galacturonates were also determined to identify the substrate binding site at 1.0 and 1.15 Å resolutions, respectively. In the binary complex, one β-
D-galactopyranuronate, Gal
pA, was found in the reducing end side of Asp153, Asp173 and Asp174, which are considered as candidates of catalytic residues. This reveals that the position of Gal
pA is the +1 subsite, thus proving the strong affinity of the +1 subsite expected from the bond cleavage frequency on oligo-galacturonates. In the ternary complex, an additional β-
D-galactofuranuronate was found in the -1 subsite. In both subsites, the recognition of the galacturonate carboxy group is important in galacturonate binding. In the +1 subsite, the carboxy group interacts with three basic residues, His195, Arg226 and Lys228, which were conserved in all endopolygalacturonases. In the -1 subsite, the unique non-prolyl
cis-peptide bond is believed to be involved in binding the carboxy group of the substrate. Based on the structures of Gal
fA and Gal
pA bound in the ternary complex, a structural model of the di-galacturonic acid part of the substrate molecule bound in both the -1 and +1 subsites across from the catalytic residues was constructed. The di-galacturonate model structure sheds light on the catalytic mechanism. Asp173 is at the appropriate position to be a proton donor to the fissile glycosidic bond. Asp153 or Asp174 seems to act as a general base to abstract a proton from the nucleophilic water.
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