Chracterization of class III peroxidases from switchgrass

Authors: MOURAL, TIMOTHY - Washington State University; LEWIS, KEVIN - Washington State University; BARNABA, CARLO - Washington State University; ZHU, FANG - Washington State University; Palmer, Nathan - Nate; Sarath, Gautam; Scully, Erin; JONES, JEFFERY - Washington State University; VERMERRIS, WILFRED - University Of Florida; Sattler, Scott; KANG, CHULHEE - Washington State University

Submitted to: Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/13/2016
Publication Date: 11/15/2016
Publication URL: https://handle.nal.usda.gov/10113/5763069
Citation: Moural, T.W., Lewis, K.M., Barnaba, C., Zhu, F., Palmer, N.A., Sarath, G., Scully, E.D., Jones, J.P., Vermerris, W., Sattler, S.E., Kang, C. 2016. Chracterization of class III peroxidases from switchgrass. Plant Pathology. 173(1):417-433. doi:10.1104/pp.16.01426.

Interpretive Summary: ClassIII peroxidases are plant enzymes notably associated with both normal growth and defensive responses of plants to a variety of environmental stress. These enzymes catalyze the formation of the plant polymer, lignin, as well as remove toxic cellular products of metabolism induced by stress thereby protecting plant cells. In this study a switchgrass peroxidase named PviPRX9 associated with lignin formation was identified and its gene cloned. The cloned gene was used to make recombinant protein in bacteria. The bacterially-produced peroxidase was purified and used to generate protein crystals. Using techniques in X-ray crystallography, the 3-dimensional structure of the PviPRX9 peroxidase was solved. Additional experiments characterized the biochemical properties of the protein. These studies provide key biochemical and structural data to understand the function of these important proteins in switchgrass plants. This knowledge combined with physiological studies can be used to improve biomass quality and plant resistance to environmental stress in the future breeding of improved switchgrass germplasm.

Technical Abstract: Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137P-X-P-X140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.