Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli.
1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyses the final step in the biosynthesis of the plant hormone ethylene. The successful overexpression and characterization of active ACC oxidase from tomato has been achieved. PCR was used to insert the corrected cDNA coding for the tomato ACC oxidase into the pET-11a expression vector. Cloning of the resultant construct in Escherichia coli BL21(DE3)pLysE gave transformants which expressed ACC oxidase at levels greater than 30% of soluble protein under optimized conditions. When induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C the ACC oxidase expressed was less soluble and less active than when induced at 27 degrees C. The enzyme was purified to near homogeneity by a three-step chromatographic procedure. The specific activity of the purified recombinant ACC oxidase was typically 1.3-1.9 mol of ethylene/mol of enzyme per min, higher than values reported for native enzyme. Like the native enzyme it displayed a requirement for ferrous iron and ascorbate, and CO2 was an activator. The ability to discriminate between racemic diastereomers of 1-amino-2-ethyl cyclopropane-1-carboxylic acid was demonstrated. The enzyme was found to have a loose specificity for ascorbate, showing apparent preference for D-ascorbate and 5,6-O-isopropylidene L-ascorbate rather than L-ascorbate. The addition of catalase, dithiothreitol and BSA to incubation mixtures all resulted in significant increases in activity. When treated with diethylpyrocarbonate (DEPC) under mildly acidic conditions, the enzyme rapidly lost activity. Comparison of the rate of inactivation with the increase in absorbance at 240 nm gave results consistent with the modification of two to three histidine residues at the active site, although the possibility of additional modification of other nucleophilic residues cannot be excluded. Inactivation was largely prevented by the addition of substrates and ferrous iron, implying that DEPC treatment results in the modification of active-site histidines, which act as ligands for ferrous iron. CO2 offered no protection against DEPC inactivation, either in the absence or presence of substrates and/or ferrous iron.
Zhang
Z
Z
Oxford Centre for Molecular Sciences, U.K.
Schofield
C J
CJ
Baldwin
J E
JE
Thomas
P
P
John
P
P
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002245:Carbon Dioxide / Q000494:pharmacology; D002374:Catalase / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity; D014162:Transfection
D018076:DNA, Complementary; D006898:Hydroxylamines; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002245:Carbon Dioxide; D012710:Serum Albumin, Bovine; D019811:Hydroxylamine; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D007501:Iron; D002374:Catalase; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase; D004047:Diethyl Pyrocarbonate; D004229:Dithiothreitol