Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/1998
Publication Date: N/A
Citation: Interpretive Summary: Hydroperoxide lyase is an enzyme present in most plants. It promotes the formation of volatile compounds from fatty acid constituents in the plant. The products are especially noticeable when a plant has been damaged, because then the enzyme and the fatty acids can easily interact to form the volatile compounds, which contribute to the aroma of the wounded plant. These volatile products may also have a role in the defense of the plant against insects or pathogens. Therefore, it is important to know the properties of this enzyme. In this paper we have characterized hydroperox- ide lyase from the stems (hypocotyls) of sunflower. Two forms of the enzyme are present in the sunflower hypocotyl cell. One form is soluble in the cell's cytoplasm, and the other is bound to membranes, most likely chloroplast membranes. It most likely exists in a complex consisting of four subunits of the enzyme. We also showed that hydroperoxide lyase contains a heme cofactor and is a cytochrome P450-type enzyme. This was the first direct demonstration that this enzyme is a cytochrome P450 protein, although hydroperoxide lyase from bell pepper fruit was predicted to be a cytochrome P450 enzyme based on its amino acid sequence. Thus, hydroperoxide lyase joins a large class of cytochrome P450 enzymes that catalyze the incorporation or transfer of oxygen atoms into physiologically active plant constituents.
Technical Abstract: Two fatty acid hydroperoxide lyases (HPO lyase I and II) were purified to apparent homogeneity from etiolated hypocotyls of sunflower (Helianthus annuus L.) by a combination of ion-exchange, hydrophobic interaction, and gel filtration chromatography. The two HPO lyases were separated during the hydrophobic interaction chromatography step, with HPO lyase I more hydrophilic than HPO lyase II. The estimated Mr of both native HPO lyases was determined by gel-filtration to be 200,000, and SDS-PAGE in the presence of 100 mM dithiothreitol showed that the enzyme was composed of a single 53 kDa peptide, suggesting that the enzyme exists as a homotetramer in vivo. The enzyme was strongly expressed in the cotyledons and green leaves. HPO lyases I and II from hypocotyl metabolized 13-hydroperoxylino- leic acid and 13-hydroperoxylinolenic acid to the same extent, but the green leaf enzyme was more than 10-fold more active with 13-hydroperoxylin- -olenic acid than 13-hydroperoxylinoleic acid. A difference spectrum between CO-bound and CO-unbound dithionite-reduced HPO lyase I showed an absorbance maximum at 452 nm, indicating that it was a cytochrome P450-type enzyme. The activities of HPO lyase I and II were significantly inhibited by nordihydroguaiaretic acid, sulfhydryl reagents, and piperonylbutoxide, which is a cytochrome P450 inhibitor.