Reaction Mechanism Based on X-ray Crystallography at Atomic Resolution of Endopolygalacutronase I from Fungus <i>Stereum purpureum</i>

Abstract

The crystal structures of its binary complex with one D-galacturonate and its ternary complex with two Dgalacturonates were also determined to identify the substrate binding site at 1.0 and 1.15 A resolutions, respectively. In the binary complex, one -D-galactopyranuronate, GalpA, 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 GalpA 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 -Dgalactofuranuronate 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 Galf A and GalpA 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.

Purification, Characterization and Amino Acid Sequence of Endopolygalacturonases IVa and IVb from Fungus Stereum purpureum

“…This number accords with the β helical structure of EndoPG I analyzed by X ray crystallography. 9,10) The resultant Tm value from the denaturation curve for EndoPG IVb was 62 C, compared to that measured for EndoPG Ia of 79.5 C 12) (Fig. 3).…”

“…8) Part of the enzyme s catalytic mechanism has also been clarified by analysis of the crystal complex (binary and ternary) occurring in reaction between enzyme and substrate. 9,10) EndoPG I with PG activity has also been successfully expressed in Echerichia coli 11) and Pichia pastoris. 12) In such a way, characteristics of EndoPG I have been studied in some detail.…”