Purification, molecular cloning and ethylene-inducible expression of a soluble-type epoxide hydrolase from soybean (Glycine max [L.] Merr.)

A soybean protein was purified from mature dry seeds. Amino-acid sequencing of the nine internal peptides derived from this N-terminally blocked prote in showed that it has a significant similarity to the soluble epoxide hydro lases known to date. A degenerate series of 23-mer oligonucleotides with se quences corresponding to an internal region of eight amino-acid residues wa s synthesized as a probe mixture for detection of a putative epoxide hydrol ase cDNA in a developing cotyledon cDNA library. The 1332-bp cDNA obtained was found to have an open-reading frame encoding the seed epoxide hydrolase -like precursor consisting of 341 amino-acid residues, suggesting that 25 a mino-acid residues upstream from the second methionine correspond to a tran sit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase-like protein was overexpressed and purified to hom ogeneity. This recombinant protein was confirmed to exhibit its epoxidediol converting activity using styrene oxide as substrate. The V-max and K-m va lues for styrene oxide are 1.36 mu mol.min-(1).mg(-1) and 1500 mu M, respec tively. Sedimentation equilibrium experiments showed that the active form o f this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12-kb genomic clone was selected and the sequence of a 1933-bp f ragment from this clone was found to cover the entire coding region togethe r with 5'- and 3'-flanking regions of the soybean epoxide hydrolase gene. T he coding region of the gene, interrupted by two short introns, was identic al to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early sta ge (13 days after flowering) and then the level of expression gradually dec reased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up-regulated by ethyl ene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcri pt was detected at significant levels and was also up-regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even tho ugh they were treated with the phytohormone. Overall, the results obtained may indicate that soluble-type epoxide hydrolase mRNA is expressed at the m aximum level in an early stage of seed development. Later, oil bodies are f ormed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydr olase transcript in some tissues in response to ethylene also indicates tha t this epoxide hydrolase may play a crucial role in self-defense systems of plant.