期刊
PHYTOCHEMISTRY
卷 94, 期 -, 页码 45-52出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.phytochem.2013.05.016
关键词
Ascorbate; Peroxidase; Panicum virgatum; Poaceae; Mutant; ABTS
资金
- USDA-ARS CRIS project [5440-21000-030-00D]
- Office of Science (BER), U. S. Department of Energy [DE-AI02-09ER64829]
- UNK Office of Graduate Studies and Research/Research Services Council
- NIH from the INBRE Program of the National Center for Research Resources [1 P20 RR16469]
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 indicates that switchgrass encodes several cytosolic ascorbate peroxidases with apparent varying levels of tissue expression. A major cytosolic ascorbate peroxidase was thus selected for further studies. This gene was cloned and expressed in Escherichia 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. A site-directed mutant, R172S, was made 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. (C) 2013 Elsevier Ltd. All rights reserved.
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