title: Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases.
abstract: Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.
authors.affiliation: *Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agriculture Utilization Research, Peoria, IL 61604, U.S.A.
authors.forename: Todd A
authors.initials: TA
authors.lastname: Naumann
authors.affiliation: *Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agriculture Utilization Research, Peoria, IL 61604, U.S.A.
authors.forename: Donald T
authors.initials: DT
authors.lastname: Wicklow
authors.affiliation: †Renewable Product Technology Research Unit, National Center for Agriculture Utilization Research, Peoria, IL 61604, U.S.A.
authors.forename: Neil P J
authors.initials: NP
authors.lastname: Price
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D020134:Catalytic Domain; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D010944:Plants; D013379:Substrate Specificity
chemical_list: D010455:Peptides; C011080:polyglycine; D002688:Chitinases; D010447:Peptide Hydrolases