3 Site directed mutagenesis of maize ChitB chitinase was used to replace two serine residues in its polyglycine linker region with glycines, resulting in altered substrate specificity for Bz-cmp and Es-cmp such that different glycine-glycine peptide bonds were cleaved. Bz-cmp hydrolyzed ChitB predominantly between glycines G3 and G4, whereas the Es-cmp activity was specific for the G1, G3, and G4 residues. Our initial findings demonstrated that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. Polyglycine hydrolases Es-cmp and Bz-cmp have been shown to cleave glycine–glycine peptide bonds within the polyglycine linker by MALDI-TOF/MS analysis of the product peptides released from the N-terminus. The chitin binding domain is truncated by alloform specific proteases and fungalysin proteases (arrows indicate site of cleavage for each). ![]() These domains are connected by a polyglycine linker. ChitA and ChitB chitinases from maize are composed of a small, amino-terminal chitin-binding hevein domain and a larger (22 kDa) chitinase domain. 4 A recent mass spectrometry analysis of reaction products showed that both Bz-cmp and Es-cmp-an enzyme from the sorghum pathogen Epicoccum sorghi-can selectively cleave glycine-glycine peptide bonds within a polyglycine linker of targeted chitinases. 3 The proteolytic activity of the polyglycine hydrolase Bz-cmp was first observed in diseased ears of maize that had been inoculated with Cochliobolus carbonum (syn. Cmp activity is exhibited by at least three types of proteases: fungalysin metalloproteases that cleave a glycine-cysteine bond that is conserved among plant class IV defense chitinases 1 alloform-specific proteases that distinguishing a single amino acid difference in maize ChitA alloforms 2 and polyglycine hydrolases, proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of targeted chitinases. ![]() Polyglycine hydrolases are one type of chitinase modifying protein (cmp), secreted fungal proteases that truncate plant class IV chitinases by cleaving near their amino-termini (Fig. This is the first report of a predicted β-lactamase that is an endoprotease. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β-lactamase-like region. BLAST searching of publicly available fungal genomes identified full-length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es-cmp to glycine, resulting in loss of catalytic activity. ![]() Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS-PAGE and MALDI-MS based assays. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. From this analysis, a 642 amino acid protein containing a predicted β-lactamase catalytic region of 280 amino acids was identified. Peptides from a tryptic digest of Es-cmp were analyzed by LC-MS/MS and the spectra obtained were matched to a draft genome sequence of E. Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. ![]() Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochliobolus carbonum (Bz-cmp). Polyglycine hydrolases are secreted fungal proteases that cleave glycine–glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases.
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