TY - JOUR
T1 - High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays
AU - Øbro, Jens
AU - Sørensen, Iben
AU - Derkx, Patrick
AU - Madsen, Christian Toft
AU - Drews, Martin
AU - Willer, Martin
AU - Mikkelsen, Jørn D.
AU - Willats, William George Tycho
N1 - Keywords
Carbohydrate microarray • Erwinia chrysanthemi • Pectin methylesterase
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
U2 - 10.1002/pmic.200800349
DO - 10.1002/pmic.200800349
M3 - Journal article
C2 - 19333997
SN - 1862-7595
VL - 9
SP - 1861
EP - 1868
JO - Proteomics - Practical Proteomics
JF - Proteomics - Practical Proteomics
IS - 7
ER -
