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Structure and disulfide bonding pattern of the hevein-like peptide domains from plant class IV chitinases

Neil P.J. Price, Frank A. Momany, Udo Schnupf, Todd A. Naumann
Physiological and molecular plant pathology 2015 v.89 pp. 25-30
Arabidopsis thaliana, Fusarium, Zea mays, alkylation, chitinase, corn, cysteine, disulfide bonds, hevein, ions, matrix-assisted laser desorption-ionization mass spectrometry, models, pathogens, peptides, protein conformation, proteinases, proteolysis
Corn (Zea mays) and Arabidopsis (Arabidopsis thaliana) produce GH family 19 plant class IV chitinases. These chitinases contain two domains: a small N-terminal hevein region, and a C-terminal chitinase. Numerous structures of GH19 chitinase domains have been reported, including the chitinase domain of corn ChitA. Structural information on the N-terminal domains, however, is lacking. Fusarium pathogens secrete fungalysin proteases that cleave some class IV chitinases at a well-defined Gly-Cys site between the two domains. To study the structure of the peptide domain we used the fungalysin protease Fv-cmp as a tool to release the hevein domain from plant class IV chitinases, allowing their direct study. MALDI-TOF MS analysis of fungalysin-released peptides from plant class IV chitinases from corn and Arabidopsis allowed visualization of multiple isotopomers, resulting in accurate mass determination. When treated with DTT, peptide ions increased in mass by six mass units, suggesting breakage of three disulfide bonds. When reduced peptides were S-alkylated, peptides were converted to a series of evenly spaced ions with various states of alkylation, confirming the presence of six reduced cysteines. This chemical data was complemented by use of molecular modeling to determine the fold of the peptide and location of disulfide bonds. The chemical data and molecular model combine to create a structural model of a hevein domain from a GH19 chitinase.