Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: Improving bioenergy and forage plants and production systems for the central U.S.

Location: Grain, Forage & Bioenergy Research

Title: Abolishing activity against ascorbate in a cytosolic ascorbate peroxidase from switchgrass

Authors
item Kovacs, Frank -
item Sarath, Gautam
item Woodworth, Kyle -
item Twigg, Paul -
item Tobias, Christian

Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 10, 2013
Publication Date: June 26, 2013
Repository URL: http://handle.nal.usda.gov/10113/57903
Citation: Kovacs, F., Sarath, G., Woodworth, K., Twigg, P., Tobias, C.M. 2013. Abolishing activity against ascorbate in a cytosolic ascorbate peroxidase from switchgrass. Phytochemistry. doi 10.1016/j.phytochem.2013.05.016.

Interpretive Summary: All plants contain enzymes and small compounds that help to detoxify cells of harmful oxidizing chemicals, such as hydrogen peroxide. One such cellular system, uses ascorbic acid (vitamin C) and the enzyme ascorbate peroxidase (APx) to convert hydrogen peroxide into water. Key amino acid residues in an enzyme can confer activity against specific substrates, and these residues can inform about the how an enzyme functions. In this study, we have made switchgrass APx in bacterial cells and specifically mutated a key amino acid to observe if we could abolish activity against vitamin C. Our data indicates that the mutant enzyme was unable to utilize ascorbate as a substrate, but could still function as a peroxidase.

Technical Abstract: Switchgrass (Panicum virgatum L.) is being developed as a bioenergy species. Recently an early version of its genome has been released permitting a route to the cloning and analysis of key proteins. Ascorbate peroxidases (APx) are an important part of the antioxidant defense system of plant cells and present a well studied model to understand structure-function relationships. Analysis of the genome revealed that switchgrass encodes several cytosolic APxs with apparent varying levels of tissue expression. We selected a major cytosolic ascorbate peroxidase for further studies. This gene was cloned and expressed in E.coli cells to obtain purified active protein. Full heme incorporation of the enzyme was achieved utilizing slow growth and supplementing the media with 5-aminolevulinic acid. The enzyme was observed to be monomeric in solution via size exclusion chromatography. Activity toward ascorbate was observed that was non-Michaelis-Menten in nature. We made a site-directed mutant, R172S in an attempt to differentiate activity against ascorbate versus other substrates. The R172S protein exhibited negligible ascorbate peroxidase activity, but showed near wild type activity toward other aromatic substrates.

Last Modified: 9/2/2014
Footer Content Back to Top of Page