1.68-Å Crystal Structure of Endopolygalacturonase II fromAspergillus niger and Identification of Active Site Residues by Site-directed Mutagenesis

Abstract

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel ␤-helix with 10 complete turns. The ␤-helix is composed of four parallel ␤-sheets, and has one very small ␣-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the ␤-helix. The plant cell wall consists of a network of complex carbohydrates like cellulose, hemicellulose, and pectin. The latter is the most complex of these carbohydrates. It contains so-called "smooth regions" and "hairy regions." The smooth regions, also known as homogalacturonan, consist of ␣(1,4)-linked D-galacturonic acid residues, whereas the hairy regions, or rhamnogalacturonan I, are characterized by stretches of alternating ␣(1,2)-linked D-galacturonic acid and L-rhamnose (1). The rhamnose residues can be substituted at their O4 atoms by arabinose or galactose (2). Throughout the pectin molecule, the galacturonic acid residues can be methylated at O6 and/or acetylated at O2 and/or O3 (3). Due to its complex structure, modification of pectin by plants or complete breakdown by microorganisms requires many different enzymes.In microorganisms several classes of pectinases have been identified. These classes comprise pectate-, pectin-, and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases, and polygalacturonases, which all depolymerize the main chain; and pectin methylesterases and pectin-and rhamnogalacturonan acetylesterases, which act on the substituents of the main chain. Crystal structures are known of members of several classes of main chain depolymerizing pectinases. These include pectate lyases from Erwinia chrysanthemi and Bacillus subtilis (4 -6), pectin lyases from Aspergillus niger (7,8), and rhamnogalacturonase A from Aspergillus aculeatus (9). Recently, the crystal structure of endopolygalacturonase from the bacterium Erwinia carotovora was solved (10). The lyases cleave the substrate by ␤-elimination, whereas rhamnogalacturonases and polygalacturonases use acid/base-catalyzed hydrolysis (11,12). Despite their completely different reaction mechanisms, and their groupings in different sequence homology families, the x-ray structures of pectate lyase, pectin lyase, and rhamnogalacturonase reveal a similar unique right-handed parallel ␤-helix topology (13,14).

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

“…We have targeted several residues of FmPG that are conserved in all of the known fungal PGs. Among them, Asp-191, Asp-212, Asp-213, Arg-267, and Lys-269 are located, as in AnPGII, inside the deep cleft (35) and form the putative active site (Fig. 1, Fig.…”

“…Given their position in proximity of the nucleophilic water molecule but at opposite sides, and their relative distance of 4.06 Å, Arg-267 and Lys-269 putatively bind the substrate at subsites ϩ1 and Ϫ1, respectively (where ϩ1 and Ϫ1 are the residues at the reducing and non-reducing ends, respectively; ref. 35).…”

“…The determined structures include those of pectin and pectate lyases, which cleave pectin and pectate by a ␤-elimination mechanism (33, 37), of a rhamnogalacturonase, which acts on the ''hairy region'' of pectin by hydrolyzing the glycosydic bond between galacturonic acid and rhamnose (38), and of two polygalacturonases, one from the bacterium Erwinia carotovora (34) and one from the fungus A. niger (AnPGII) (35). With our determination of the x-ray structure of the PG from F. moniliforme (FmPG), we have now complete structural data about two fungal polygalacturonases, the structure of FmPG being the first one from a true phytopathogenic fungus.…”

“…From the the crystal structure of FmPG we can infer its mode of action. FmPG can be classified into the family 28 of glycosyl hydrolases (39) and, given its structural similarity to the AnPGII, it is likely to be an inverting enzyme with a non-conventional mechanism of action (35). A single displacement reaction may be catalyzed where an acidic residue acts as a general acid (Asp212) by donating a proton to the glycosydic oxygen, and residues Asp-191 and Asp-213 activate a water molecule, which performs a nucleophilic attack on the anomeric carbon.…”

A Novel Structural Fold in Polysaccharide Lyases

“…The structure and function relationships of polygalacturonan-degrading enzymes, including hydrolases and lyases have also been well documented (13)(14)(15)(16)(17)(18)(19)(20). However, little information is available on the degradation of RG regions by microbes except for Aspergillus species (21) and Erwinia chrysanthemi (22,23).…”