12-Oxophytodienoate reductases (OPRs) belong to a family of flavin-dependent oxidoreductases. With two new tomato isoforms reported here, three OPRs have now been characterized in both tomato and Arabidopsis. Only one of these isoforms (OPR3) participates directly in the octadecanoid pathway for jasmonic acid biosynthesis, as only OPR3 reduces the 9S,13S-stereoisomer of 12-oxophytodienoic acid, the biological precursor of jasmonic acid. The subcellular localization of OPRs was analyzed in tomato and Arabidopsis. The OPR3 protein and activity were consistently found in peroxisomes where they co-localize with the enzymes of beta-oxidation which catalyze the final steps in the formation of jasmonic acid. The octadecanoid pathway is thus confined to plastids and peroxisomes and, in contrast to previous assumptions, does not involve the cytosolic compartment. The expression of tomato (Lycopersicon esculentum,Le) OPR3 was analyzed in the context of defense-related genes using a microarray comprising 233 cDNA probes. LeOPR3 was found to be up-regulated after wounding with induction kinetics resembling those of other octadecanoid pathway enzymes. In contrast to the induction of genes for wound response proteins (e.g. proteinase inhibitors), the accumulation of octadecanoid pathway transcripts was found to be more rapid and transient in wounded leaves, but hardly detectable in unwounded, systemic leaves. Consistent with the expression data, OPDA and JA were found to accumulate locally but not systemically in the leaves of wounded tomato plants. The transcriptional activation of the octadecanoid pathway and the accumulation of JA to high levels are, thus not required for the activation of defense gene expression in systemic tissues.

Background: 12-Oxophytodienoate reductase (OPR) is a flavin mononucleotide (FMN)-dependent oxidoreductase in plants that belongs to the family of Old Yellow Enzyme (OYE). It was initially characterized as an enzyme involved in the biosynthesis of the plant hormone jasmonic acid, where it catalyzes the reduction of the cyclic fatty acid derivative 9S,13S-12-oxophytodienoate (9S,13S-OPDA) to 1S,2S-3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoate. Several isozymes of OPR are now known that show different stereoselectivities with regard to the four stereoisomers of OPDA.Results: Here, we report the high-resolution crystal structure of OPR1 from Lycopersicon esculentum and its complex structures with the substrate 9R,13R-OPDA and with polyethylene glycol 400. OPR1 crystallizes as a monomer and folds into a (beta alpha)(8) barrel with an overall structure similar to OYE. The cyclopentenone ring of 9R,13R-OPDA is stacked above the flavin and activated by two hydrogen bonds to His187 and His190. The olefinic bond is properly positioned for hydride transfer from the FMN N(5) and proton transfer from Tyr192 to C beta and C alpha, respectively. Comparison of the OPR1 and OYE structures reveals striking differences in the loops responsible for binding 9R,13R-OPDA in OPR1.Conclusions: Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that alpha,beta -unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9R,13R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9R,13R-OPDA or a structurally related oxylipin as natural substrate of OPR1.

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed inEscherichia coli in N-terminal fusion with glutathioneS-transferase and was purified from bacterial extracts.LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K m (15.2 ± 2.4 μm) andK m /k cat (0.94 ± 0.11 mm −1 × s−1) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate.LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl2 and to some extent also by Mg2+, Ca2+, and Co2+, whereas other divalent metal ions (Cu2+, Zn2+) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P.

A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Fellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) from Arabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein in Escherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest with N-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of the LeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone, Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration with para-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR.