title: High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays.
abstract: Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray-based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.
authors.affiliation: Department of Biology, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Øbro
authors.forename: Iben
authors.initials: I
authors.lastname: Sørensen
authors.forename: Patrick
authors.initials: P
authors.lastname: Derkx
authors.forename: Christian T
authors.initials: CT
authors.lastname: Madsen
authors.forename: Martin
authors.initials: M
authors.lastname: Drews
authors.forename: Martin
authors.initials: M
authors.lastname: Willer
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
authors.forename: William G T
authors.initials: WG
authors.lastname: Willats