Thesis Title: Biophysical studies of an important processive enzyme of the plant cell wall.
A host of plant pectin methylesterases (PMEs) modify the complex polysaccharide pectin, a key component of the plant cell wall. PMEs play critical roles in all plant tissues and at every stage of the plant life cycle. Fungal and bacterial phytopathogens, phytophagous beetles and weevils, and human-gut pathogens and commensals, also express PMEs to attack the plant cell well. PMEs modify the overall amount and distribution of methylester groups on pectin in a variety of ways, yielding charged groups that can be arranged randomly (resulting from a non-processive mode of action) or in a blockwise fashion (arising from processive processing): always ensuring that the polysaccharide patterning is optimised spatiotemporally for its biological purpose. We propose to build on the insights obtained from our pioneering molecular dynamics (MD) studies of a processive bacterial PME, our experimental expertise honed in the first structural study of a non-processive fungal PME and recent experience with single molecule experiments to learn how processivity is controlled. Our results augment studies on processive plant and bacterial PMEs, and further work promises not only to provide a raison d’être for the copious number of PME isoforms that exist, but to illuminate routes for designing novel architectures of this commercially important biopolymer.
PMEs hydrolyse ester groups of the homogalacturonan (HG) backbone of pectin, releasing methanol and exposing negatively charged carboxylate groups on the pectin, altering its properties and those of the plant cell wall. In addition to processive-to-non-processive modes of action, some PMEs operate at near-neutral to basic pH, others at acidic pH; some require electrolyte (e.g. NaCl) for activity, others do not. We understand little of the subtleties of this deceptively simple reaction, especially of pectin recognition.
We aim combine functional and X-ray structural studies on PMEs (and mutants) with molecular dynamics simulations and single-molecule experiments to ‘see’ this enzyme in action. Ultimately, we seek to use the knowledge gained to design PME-modified pectins, for example with specific patternings, endowing pectin with new functionalities for applications in a variety of contexts, including the food industry, where pectin and pectin methylesterases are multi-billion dollar ingredients and products.