Heterologous Expression and Characterization of Soybean Cytosolic Ascorbate Peroxidase

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to...

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Bibliographic Details
Published in:	Archives of biochemistry and biophysics Vol. 328; no. 1; pp. 1 - 8
Main Authors:	Dalton, David A., del Castillo, Leonor Diaz, Kahn, Michael L., Joyner, Shannon L., Chatfield, J.Mark
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-04-1996
Subjects:	ACIDE ASCORBIQUE ACIDO ASCORBICO ACTIVIDAD ENZIMATICA ACTIVITE ENZYMATIQUE ADN ADN RECOMBINADO ADN RECOMBINE Amino Acid Sequence Apoenzymes - biosynthesis Apoenzymes - genetics Apoenzymes - isolation & purification Apoenzymes - metabolism ascorbate peroxidase Ascorbate Peroxidases Base Sequence Cell Compartmentation CLONACION CLONAGE Cloning, Molecular Coenzymes - biosynthesis Coenzymes - genetics Coenzymes - isolation & purification Coenzymes - metabolism Cytosol - enzymology ESCHERICHIA COLI Escherichia coli - genetics GLYCINE MAX Glycine max - enzymology Glycine max - genetics Histidine - genetics MINERALOGIA MINERALOGIE Molecular Sequence Data nitrogen fixation NODOSITE RACINAIRE nodule NUDOSIDADES RADICULARES PEROXIDASAS Peroxidases - biosynthesis Peroxidases - genetics Peroxidases - isolation & purification Peroxidases - metabolism PEROXYDASE PURIFICACION PURIFICATION Recombinant Proteins - biosynthesis Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism soybean (Glycine max) Spectrophotometry ascorbate peroxidase nodule nitrogen fixation soybean (Glycine max)
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Summary:	Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system inEscherichia coli.The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni2+-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of δ-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form byin vitroreconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.
Bibliography:	9615017 F60 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-9861 1096-0384
DOI:	10.1006/abbi.1996.0135