Molecular basis of the activity of the phytopathogen pectin methylesterase

Abstract

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.

Figure 1

Substrate binding to PME. (A) Stereoview of the cartoon representation of the three dimensional structure of PME with bound hexasaccharide (compound II) shown in ‘stick' representation. Residues D199, R267 and D178A are also shown. The mutation D178A mutant was used to trap the Michaelis complex. (B) Surface representation of PME together with stick representation of hexasacharide II showing the shape and position of the substrate-binding cleft, the reducing end of the hexasaccharide is to the right of this Figure and the substrate would move through the binding site from right to left. (C) The four specifically methylated hexasaccharides used in this work. Compound I: R1=R2=Me, R3=H; compound II: R1=R2=H, R3=Me; compound III: R1=Me, R2=R3=H; compound IV: R1=H, R2=R3=Me. Pymol (DeLano, 2002) was used to produce this figure and also Figures 2 and 3.

Figure 2

Enzyme–substrate interactions in detail. (A) Stereo-view of the stick model of the Michaelis complex formed using compound II overlaid with maximum likelihood/σ_A weighted 2F_o−F_c syntheses contoured at 1.5 σ and (B) schematic diagram showing the interactions involved. (B) was produced using LIGPLOT (Wallace et al, 1995).

Figure 3

Stereo-view of the active site residues in the Michaelis complex formed using compound II and the D178A mutant of PME. Asp 178 (the general acid-base) is built back into this structure in the conformation seen in the wild-type enzyme, the conformation of the other residues and substrate are as seen in the electron density map of the complex.

Figure 4

The proposed mechanism of PME based on the crystal structures and kinetic analyses of directed mutants.