Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli

Abstract

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.

Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene

“…The enzyme obtained from this purification protocol (see Table 1) had a specific activity in the range of 2.3-3.1 mol of ethylene per min per mol of ACCO, which is comparable to other reports of recombinant ACCO purification protocols (15).…”

“…The [ 15 N Ϫ 14 N] difference spectrum (Fig. 2, trace D) reveals the gain of a doublet from a single 15 . Such large hyperfine interactions require that the amino group of alanine be directly coordinated to the Fe-NO center of the Fe(II)ACCO⅐alanine⅐NO adduct.…”

“…E͞D values were determined by using RHOMBO ⁄2) each peak of the doublet is further split into 2I lines, each separated by 3P, with P being the quadrupole coupling parameter. The -feature is often absent in Q-band ENDOR spectra (15). An ENDOR experiment performed at the low-and high-field edges (''single-crystal like'' positions) of an EPR signal interrogates only a single orientation, or subpopulation; at intermediate fields a well-defined subset of orientations is examined; analysis of such ''orientationselective'' spectra can permit complete analysis of hyperfine and quadrupole interaction tensors (18).…”

Identification of Strictly Conserved Histidine and Arginine Residues as Part of the Active Site in Petunia hybrida Flavanone 3β‐Hydroxylase

“…Moreover, it seems that ascorbate is able to bind to FHT even in the absence of Fez+ and to protect the enzyme from chemical modification at the active site histidines. Whereas for ACC oxidase no protective effect was found by the addition of ascorbate [41], a similar phenomenon as described for FHT was observed for prolyl 4-hydroxylase (141. These results, as well as the observed low Michaelis constant of FHT for ascorbate and the loss of enzyme activity in the absence of ascorbate, confirm the important role of this soluble cofactor.…”

“…Indirect evidence for the involvement of histidines in the catalytic mechanism of FHT and other 2-oxoglutarate-dependent dioxygenases as well as the related non-heme iron enzymes, has been reported in recent studies on the inhibition of these enzymes by diethylpyrocarbonate [6,14,41,421. For prolyl 4-hydroxylase, experiments with cofactors as protecting agents against inactivation by diethylpyrocarbonate demonstrated that the best protection can be observed when 2-oxoglutarate, ferrous ions, and ascorbate were present simultaneously [14].…”