|CHAUDET, MARCIA - University Of Waterloo|
|ROSE, DAVID - University Of Wisconsin|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/27/2017
Publication Date: N/A
Technical Abstract: Polyglycine hydrolases are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn. Polyglycine hydrolases are novel proteins in terms of activity and sequence. The objective of the study is to understand how they recognize their substrates and cleave featureless polyglycine sequences. Multiple proteases, plant chitinase substrates, and mutant forms of each were produced recombinantly. SDS-PAGE and MALDI-TOF/MS based in vitro protease assays were performed. We identified the catalytic serine within an SXXK motif, indicating that the active site is related to that of bacterial beta-lactamases. Analysis of reaction products from assays with plant chitinases and analog peptides showed that this active site plays a limited role in substrate specificity. Through mutagenesis studies we have found that seven invariant tryptophans are crucial for polyglycine hydrolase folding and stability. We also found protease mutations that change cleavage site preference. In summary, the activity of these novel proteases has been characterized and we are mapping these activities to the amino acid sequence.