Biochemical characterization and molecular insights into substrate recognition of pectin methylesterase from Phytophthora infestans

Abstract

Pectin methylesterases (PMEs) are a class of carbohydrate-active enzymes that act on the O6-methyl ester groups of the homogalacturonan component of pectins, resulting in de-esterification of the substrate polymers and formation of pectate and methanol. PMEs occur in higher plants and microorganisms, including fungi, oomycetes, bacteria, and archaea. Microbial PMEs play a crucial role in pathogens' invasion of plant tissues. Here, we have determined the structural and functional properties of Pi-PME, a PME from the oomycete plant pathogen Phytophthora infestans. This enzyme exhibits maximum activity at alkaline pH (8.5) and is active over a wide temperature range (25-50 °C). In silico determination of the structure of Pi-PME reveals that the protein consists essentially of three parallel β-sheets interconnected by loops that adopt an overall β-helix organization. The loop regions in the vicinity of the active site are extended compared to plant and fungal PMEs, but they are shorter than the corresponding bacterial and insect regions. Molecular dynamic simulations revealed that Pi-PME interacts most strongly with partially de-methylated homogalacturonans, suggesting that it preferentially uses this type of substrates. The results are compared and discussed with other known PMEs from different organisms, highlighting the specific features of Pi-PME.

Keywords: Molecular simulation; Oomycete; Pectin methylesterases; Phytophthora infestans; Potato late blight.

Graphical abstract

Fig. 1

2D representation of the three different pentamers of α-(1–4)-linked d-galacturonosyl residues used for modelling and simulation studies.

Fig. 2

Evolutionary sequence conservation analysis of the PME family. The red rectangular boxes highlight the conserved key active site residues and the green boxes indicate conserved sequence motifs.

Fig. 3

Phylogenetic analysis of 99 representative PME sequences from different taxa. Note: red * represents Pi-PME; green * represents PME from the plant Vitis riparia.

Fig. 4

Determination of the optimal temperature (a) and optimal pH (b), of activity of Pi-PME.

Fig. 5

(a) Model of the 3D structure of Pi-PME (violet) superimposed on the template structure of A. niger (orange) and D. carota (green), (b) Structural superposition of other PMEs structure; loops region variations are highlighted in voilet (Rice Weevil PME); green (PME from D. dadantii); and red (Pi-PME).

Fig. 6

An average structure of Pi-PME obtained after MD simulation showing two extreme conformations of the flexible loop I region (red and blue), (b) RMSD vs time evolution of the entire Pi-PME protein (light blue) and loop I region (magenta).

Fig. 7

MD simulation of complexes of Pi-PME and three different homo-oligogalacturonan substrates.

Fig. 8

(a) Conformations of the substrate obtained from different time intervals (0–200 ns) during the entire simulation, (b) 2D representation of the substrate-binding mode in the active site of Pi-PME.

Fig. 9

Key active site residues involved in the catalytic mechanism and their distance from the methylester group of the substrate during MD simulation.