Heterologous expression and site-directed mutagenesis of the 1-aminocyclopropane-1-carboxylate oxidase from kiwi fruit.

A system has been developed for the expression in Escherichia coli of 1-aminocyclopropane-1-carboxylate (ACC) oxidase from kiwi fruit. In this first report of site-directed mutagenesis of ACC oxidase, seven different mutants of the enzyme have been expressed, and their activities compared to that of the heterologoulsy expressed wild-type enzyme. No great loss of activity was observed when Lys172 was substituted by either Ala or Cys, or when Gly137 was substituted by Pro. However, the mutant proteins showed only 1% of the wild-type activity when substitutions were made of Asp179, His177, and Lys158. The results are discussed in terms of possible mechanisms by which ACC oxidase is activated by carbon dioxide, and in terms of structural motifs suggested by the known structure of isopenicillin N-synthase, an enzyme related by mechanism and sequence similarity to ACC oxidase. It is concluded that Lys172, a putative carbon dioxide binding site, has no role to play in the catalytic activity of the enzyme. The results support a previous suggestion that ACC oxidase shares important structural features with isopenicillin N-synthase.

Characterization of the safener-induced glutathione S-transferase isoform II from maize.

The safener-induced maize (Zea mays L.) glutathione S-transferase, GST II (EC 2.5.1.18) and another predominant isoform, GST I, were purified from extracts of maize roots treated with the safeners R-25788 (N,N-diallyl-2-dichloroacetamide) or R-29148 (3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidone). The isoforms GST I and GST II are respectively a homodimer of 29-kDa (GST-29) subunits and a heterodimer of 29- and 27-kDa (GST-27) subunits, while GST I is twice as active with 1-chloro-2,4-dinitrobenzene as GST II, GST II is about seven times more active against the herbicide, alachlor. Western blotting using antisera raised against GST-29 and GST-27 showed that GST-29 is present throughout the maize plant prior to safener treatment. In contrast, GST-27 is only present in roots of untreated plants but is induced in all the major aerial organs of maize after root-drenching with safener. The amino-acid sequences of proteolytic fragments of GST-27 show that it is related to GST-29 and identical to the 27-kDa subunit of GST IV.

Cloning and characterization of maize herbicide safener-induced cDNAs encoding subunits of glutathione S-transferase isoforms I, II and IV.

Several GSTs have been characterised in maize. GST I is a homodimer of 29 kDa subunits, GST II a hetrodimer of 27 kDa and 29 kDa subunits and GST IV a homodimer of 27 kDa subunits. We report the isolation and characterization of a herbicide-safener inducible cDNA clone, GST-27. Based on partial amino acid sequence, GST-27 encodes the 27 kDa subunit present in both glutathione S-transferase isoforms GST II and IV. Northern blotting was used to compare the expression patterns of GST-27 with that of GST-29. Transcripts corresponding to GST-27 were found to be constitutively expressed in RNA isolated from the root, but no expression was detected in RNA isolated from aerial parts of the plant. The application of herbicide safener caused a dramatic increase in the expression of GST-27 in all aerial plant parts tested. GST-29 was found to be constitutively expressed in RNA isolated from a number of maize tissues. The basal level of GST-29 expression showed a minimal increase upon herbicide safener treatment. Although a range of hormonal, environmental and physiological stimuli failed to elevate GST-27 levels, some increase in GST-27 mRNA was observed in the late stages of leaf senescence and after treatments resulting in phytotoxic effects.