Molecular characterization of NbEH1 and NbEH2, two epoxide hydrolases from Nicotiana benthamiana

Abstract

Plant epoxide hydrolases (EH) form two major clades, named EH1 and EH2. To gain a better understanding of the biochemical roles of the two classes, NbEH1.1 and NbEH2.1 were isolated from Nicotiana benthamiana and StEH from potato and heterologously expressed in Escherichia coli. The purified recombinant proteins were assayed with a variety of substrates. NbEH1.1 only accepted some aromatic epoxides, and displayed the highest enzyme activity towards phenyl glycidyl ether. In contrast, NbEH2.1 displayed a broad substrate range and similar substrate specificity as StEH. The latter enzymes showed activity towards all fatty acid epoxides examined. The activity (Vmax) of NbEH1.1 towards phenyl glycidyl ether was 10 times higher than that of NbEH2.1. On the contrary, NbEH2.1 converted cis-9,10-epoxystearic acid with Vmax of 3.83μmolminmg(-1) but NbEH1.1 could not hydrolyze cis-9,10-epoxystearic acid. Expression analysis revealed that NbEH1.1 is induced by infection with tobacco mosaic virus (TMV) and wounding, whereas NbEH2.1 is present at a relatively constant level, not influenced by treatment with TMV and wounding. NbEH1.1 transcripts were present predominantly in roots, whereas NbEH2.1 mRNAs were detected primarily in leaves and stems. Overall, these two types of tobacco EH enzymes are distinguished not only by their gene expression, but also by different substrate specificities. EH1 seems not to participate in cutin biosynthesis and it may play a role in generating signals for activation of certain defence and stress responses in tobacco. However, members of the EH2 group hydrate fatty acid epoxides and may be involved in cutin monomer production in plants.