soft-rot disease in a variety of crops (8). The pelC gene has been cloned into Escherichia coli, with the latter constructs exhibiting maceration activity comparable to E. chrysanthemi (9). By using the recombinant form from E. chrysanthemi, the threedimensional structure of PelC has been determined to fold into a novel topology, not observed before 1993 or predicted during the first 30 years of crystallography (10, 11). PelC folds into a large right-handed coil termed the parallel β helix. There are eight coils in the helix, and each is comprised of three β strands, connected by three turns with unique features. When the coils are stacked, the structure has the appearance of three parallel β sheets, stabilized by an extensive network of interstrand hydrogen bonds. Another notable feature of the PelC fold is the internal organization of the amino acids that form the core of the parallel β helix. All of the amino acids, which are oriented toward the interior, are regularly aligned with amino acids from neighboring coils, giving rise to long ladders of hydrophobic, aromatic, or polar amino acids. In contrast, the exterior amino acids are randomly oriented and comprise loops, of varying length and composition, which protrude from the central core. The initial postulate that the protruding loops form the substrate and active site has proven to be correct, as will be discussed in a later section. From the perspective of an effective plant virulence factor, the most important feature of the parallel β helix fold is the stability that it confers upon an enzyme that must function in the hostile extracellular environment.
Most enzymes that degrade the plant cell wall are members of the glycoside hydrolase superfamily. Of the 62 families, organized according to sequence similarities, three-dimensional structures for members of 28 families have been reviewed (12). The enzymes that degrade the cellulose network of the plant cell wall are represented by a diversity of protein folds. The predominant structural fold, represented by endoglucanases and xylanases, is the (β/α)8 barrel. In this topology, eight parallel β strands, arranged in a circular, barrel-like motif, are connected by α helices that cover the exterior of the β barrel. The polysaccharide substrate site is located at the carboxy terminal base of the barrel, with the catalytic residues often found on β strands 4 and 7 (13). The structures of other cellulases fold into antiparallel β motifs as well as into domains comprised entirely of α helices. For example, cellobiohydrolase folds into a distorted antiparallel β barrel, sometimes called a β sandwich (14). Loops of different lengths and conformations fold over one face of the β sandwich and form the substrate-binding tunnel. Another example is endoglucanase CelD from Clostridium thermocellum, a cellulase that is structured into a motif best described as an (α/α)6 barrel (15). In contrast to the cellulase and hemicellulase families, the enzymes that degrade the pectate network all share the same parallel β helix topology initially found in PelC (Fig. 1). These include two additional pectate lyases, E. chrysanthemi PelE (16), Bacillus subtilis Pel (17); two pectin lyases from Aspergillus niger, PLA (18) and PLB (19); one rhamnogalacturonase, Aspergillus aculeatus RGase A (20); and two polygalacturonases, Erwinia carotovora polygalacturonase (21) and A. niger endopolygalacturonase II (22). Although the mechanism of pectic cleavage differs for the hydrolases and the lyases, the substrate binding sites are all found in a similar location within a cleft formed on the exterior of the parallel β helix. Moreover, parallel β helix folds, with analogous substrate-binding clefts, have been found in enzymes that degrade oligosaccharides found on cell surface receptors, such as P22 tailspike endorhamnosidase (23), and in the intercellular matrix, such as chondroitinase B (24).
Before 1990, publication of a plethora of biochemical data usually preceded the publication of a three-dimensional structure. The two types of information are mutually beneficial. A structure explains the biochemical data and the biochemical data helps to elucidate the active site of an enzyme or the functionally relevant parts of a protein. However, in the last decade, threedimensional structures have been determined at such a rapid pace, as a consequence of dramatically improved x-ray diffraction and NMR techniques, that it is now more common to solve a structure, in the absence of any supporting biochemical data. Thus, new strategies are being developed to extract functional information from static three-dimensional images. PelC has been among the first group of protein structures solved, without the benefit of relevant biochemical data to interpret the functional aspects of the three-dimensional structure. When the PelC structure was reported, details of its enzymatic mechanism could be summarized in the following few sentences. PelC is composed of 353 amino acids, with a molecular mass of 37,676 daltons and two disulfide bonds (9). As shown in Fig. 2, PelC catalyzes the endo- and exolytic cleavage of the α-1-4 glycosidic bond of polygalacturonic acid, generating an unsaturated trimer end product (25). Ca2+ is essential for the in vitro reaction and is likely required in the in vivo reaction in the plant cell wall in which the Ca2+ concentration is estimated to be as high as 1.0 mM. PelC lyase activity can be detected from a pH of 6.2 to a pH of 11.2, with an optimal pH at 9.5 (26). No amino acids, which participate in catalysis or substrate binding, had been identified by classical techniques. Consequently, neither the active site nor the substrate-binding pocket could be deduced with certainty from the initial PelC structure. The native PelC structure does provide two clues about the location of the region involved in catalysis (10). First, a Ca2+-like, heavy atom derivative binds in