Purification and characterization of a lentil seedling lipoxygenase expressed in E. coli: Implications for the mechanism of oxodiene formation by lipoxygenases

Abstract

Lentil seedlings contain at least six lipoxygenase isoenzymes, which are difficult to separate by classical enzyme purification techniques. The aim of this work was to study one particular lentil seedling lipoxygenase, as previous work indicated possible interesting characteristics of this enzyme with respect to oxodiene formation. Since it proved to be difficult to obtain this enzyme in significant quantities in a pure state, we expressed it in Escherichia coli. Using an expression vector based on the T7 RNA polymerase promoter (pET11d) we achieved of a fully functional lentil seedling lipoxygenase in E. coli, which was purified to homogeneity by DEAE ion-exchange chromatography and gel-filtration. The products obtained from linoleic acid were analysed. This recombinant lipoxygenase corresponds to that found in the lower part of the epicotyl and in the hypocotyl of the lentil seedling. It produces predominantly 13-(S)-hydroperoxy-(9Z,11E)-octadecadienoic and minor amounts of 9(S)-hydroperoxy-(10E,12Z)-octadecadienoic acids, as well as significant amounts of C18-oxodienes with a regiospecificity different from hydroperoxide formation. The latter mixture was found to consist of equal amounts of 13-oxo-(9Z,11E)-octadecadienoic and 9-oxo-(10E,12Z)-octadecadienoic acids. It is concluded that (1) oxodienes formed by this lentil enzyme do not originate from a secondary conversion of hydroperoxides, but rather from a different lipoxygenase-catalysed reaction and (2) this lipoxygenase shows similarities to pea lipoxygenase G, with both representing a novel type of legume lipoxygenase.