Submitted to: Protein Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2015
Publication Date: 5/22/2015
Publication URL: http://handle.nal.usda.gov/10113/62933
Citation: Naumann, T.A., Naldrett, M.J., Ward, T.J., Price, N.P. 2015. Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases. Protein Science. 24(7):1147-57. doi: 10.1002/pro.2705.
Interpretive Summary: Polyglycine hydrolases are a type of fungal protein that attacks plant defenses. We previously discovered these proteins in rotted ears of corn from a production field and have described their activity in laboratory reactions. In this research, we determined their amino acid sequence. This is a tremendous advance. It allows production of large quantities of protein for study, the study of the relationship between the sequence and the protein's activity, comparison of the sequence to previously studied proteins, and the abililty to determine which pathogenic fungi make similar proteins. We found that a large part of the polyglycine hydrolase sequence is unlike that of any protein that has been described before, while part of these proteins is similar to bacterial proteins that destroy penicillin and related antibiotics. Highly similar protein were found in a number of plant pathogenic fungi while more distant matches extend to woodland mushrooms. This research is important because it increases our knowledge of how fungal plant pathogens cause plant diseases. The information also helps all protein scientists by matching a unique protein activity to a novel amino acid sequence.
Technical Abstract: Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochliobolus carbonum (Bz-cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. 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. sorghi. From this analysis, a 642 amino acid protein containing a predicted B-lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS-PAGE and MALDI-MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es-cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full-length homologous proteins in eleven other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the B-lactamase-like region. This is the first report of a predicted B-lactamase that is an endoprotease.