Active-Site Architecture of Endopolygalacturonase I fromStereum purpureumRevealed by Crystal Structures in Native and Ligand-Bound Forms at Atomic Resolution^,

Abstract

Crystal structures of endopolygalacturonase from Stereum purpureum were solved in native and two galacturonic acid complex states at atomic resolution. Endopolygalacturonase catalyzes the hydrolysis of alpha-1,4-glycosidic linkage of polygalacturonate in pectin. The native structure was determined by the multiple wavelength anomalous dispersion method and was refined anisotropically with SHELXL-97, with an R factor of 11.4% and an R(free) factor of 14.0% at 0.96 A resolution. The enzyme folds into a right-handed parallel beta-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 A resolutions, respectively. In the binary complex, one beta-D-galactopyranuronate was found in the +1 subsite, thus proving the strong affinity of the +1 subsite expected from the bond cleavage frequency on oligogalacturonates. In the ternary complex, an additional beta-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 nonprolyl cis-peptide bond is believed to be involved in binding the carboxy group of the substrate. The active site architecture of the complexes provides insight into the mechanism of inverting glycosyl hydrolases and also sheds light on the basis of the differences between the family 28 and the other inverting glycosyl hydrolases.

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).…”

“…The eight residues (Asn151, Asp153, Asp173, Asp 174, His195, Gly196, Arg226 and Lys228 in EndoPG IV) identified as invariant in endo and exopolygalacturonases by Kester 20) were conserved in the EndoPG IV amino acid sequence. Residue Asp173, identified as a general acid catalyst, and either residue Asp153 or Asp174, identified as general base catalyst candidates in EndoPG I, 9) were conserved in EndoPG IV. Furthermore, fourteen residues were identified in the amino acid sequence as candidates for constructing subsite +1 and −1 of EndoPG I, 9) and six cysteins forming three disulfide bonds were also conserved.…”

“…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.…”

Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

“…The three-dimensional structures of several PGs have been reported (Pickersgill et al 1998;van Santen et al 1999;Cho et al 2001;Federici et al 2001;Shimizu et al 2002;van Pouderoyen et al 2003) but only one structure of exopolygalacturonosidase (from Yersinia enterocolitica) with an accompanying explanation of the molecular determinants for its specialized activity (Abbott & Boraston 2007). Tertiary structure of exoPG or generally of any plant pectate hydrolase has not been reported yet.…”

Auto-inactivation of Stereum purpureum Proendopolygalacturonase I by C-terminal 44 Amino Acid Residues

“…11) 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. 12) We have designated the enzyme possessing the 44 residue C terminal sequence as Pro EndoPG I to distinguish it from mature EndoPG I. It is thought that Pro EndoPG I produced by this fungus is secreted to the external mycelium, and then converted to the mature type through the action of proteases secreted in a medium.…”