|CHAUDET, MARCIA - University Of Waterloo|
|NALDRETT, MICHAEL - Danforth Plant Science Center|
|ROSE, DAVID - University Of Waterloo|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/8/2015
Publication Date: 10/8/2015
Citation: Naumann, T.A., Chaudet, M.M., Bakota, E.L., Naldrett, M.J., Rose, D.R., Price, N.P. 2015. Structure and function of polyglycine hydrolases [abstract]. International Proteolysis Society.
Technical Abstract: Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglycine. Some glycine-glycine bonds are frequently cleaved, while others appear to have complete immunity. We have shown that PGHs are serine-type proteases from family S12, a protease family comprised of penicillin binding proteins involved in bacterial cell wall biosynthesis, '-lactamases that cleave '-lactam antibiotics, and D amino-peptidases. In addition to the catalytic region, PGHs have a larger, amino region of unknown function. To determine how primary sequence and secondary structure of PGHs relate to function we are applying multiple methodologies: recombinant protein production of PGH site-directed mutants and compelling homologous proteins identified from genomics; SDS-PAGE and MALDI-TOF MS based in-vitro protease assays with plant chitinase and peptide substrates; and X-ray crystallography. Our findings demonstrate that PGHs recognize plant chitinase substrates through exosite interactions, as truncated chitinases with intact polyglycines are not cleaved. Results with peptide substrates, however, demonstrate that the active site encodes some specificity as peptides as short as 14 amino acids are hydrolyzed by PGHs. Screening of a peptide library modeled after this substrate resulted in isolation of peptides that inhibit PGHs and protect plant chitinases in vitro. These peptides may be useful tools for increasing fungal disease resistance of crop plants.