|CHAUDET, MARCIA - University Of Waterloo|
|ROSE, DAVID - University Of Waterloo|
Submitted to: Protein Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2014
Publication Date: 3/15/2014
Citation: Chaudet, M.M., Naumann, T.A., Price, N.P., Rose, D. 2014. Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays. Protein Science. 23(5):586-593.
Interpretive Summary: Plant chitinases are proteins involved in defense against fungal pathogens. Previously, we identified ChitA chitinase as a protein that reduces ear rot and fungal toxin contamination of corn. In this research we utilized a technique called X-ray crystallography to determine the structure of ChitA. With the aid of a computer the structure can be visualized as a 3-dimensional model. This detailed model reveals the individual amino acids (the building blocks of proteins) in ChitA that directly interact with its substrate. This creates a direct link between the DNA sequence of corn and a chemical reaction involved in plant defense. This information is important because it will enable future studies of ChitA aimed at further understanding its biological role and how its activity can be manipulated in plants to reduce corn ear rot and mycotoxin contamination. The structure, moreover, will be publicly available to aid other scientists who study similar proteins.
Technical Abstract: Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA 'N), which lacks the hevein-like domain. ChitA 'N and a mutant form (ChitA 'N-EQ) were expressed and purified; enzyme assays showed that ChitA 'N activity was comparable to the full-length enzyme. Mutation of Glu62 to Gln (ChitA 'N-EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA 'N provided sstrong support for key roles for Glu62, Arg 177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein-like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.