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 construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-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 delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.
Dalton
D A
DA
Biology Department, Reed College, Portland, Oregon 97202, USA.
Diaz del Castillo
L
L
Kahn
M L
ML
Joyner
S L
SL
Chatfield
J M
JM
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D060387:Ascorbate Peroxidases; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D003067:Coenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry
D001051:Apoenzymes; D003067:Coenzymes; D011994:Recombinant Proteins; D006639:Histidine; D010544:Peroxidases; D060387:Ascorbate Peroxidases