Investigation of Substrate Binding and Product Stereochemistry Issues in Two Linoleate 9‐Lipoxygenases

Herein we characterize the Arabidopsis thaliana AtLOX1 and tomato ( Solanum lycopersicum ) LOXA proteins as linoleate 9 S ‐lipoxygenases (9‐LOX), and use the enzymes to test a model that predicts a relationship between substrate binding orientation and product stereochemistry. The cDNAs were heterologously expressed in E. coli and the proteins partially purified by nickel affinity chromatography using a N‐terminal (His) 6 ‐tag. Both enzymes oxygenated linoleic acid almost exclusively to the 9 S ‐hydroperoxide with turnover numbers of 300–400/s. AtLOX1 showed a broad range of activity over the range pH 5–9 (optimal at pH 6); tomato LOXA also showed optimal activity around pH 5–7 dropping off more sharply at pH 9. Site‐directed mutagenesis of a conserved active site Ala (Ala562 in AtLOX1, Ala 564 in tomato LOXA, and typically conserved as Ala in S ‐LOX and Gly in R ‐LOX), revealed that substitution with Gly led to the production of a mixture of 9 S ‐ and 13 R ‐hydroperoxyoctadecadienoic acids from linoleic acid. To follow up on earlier reports of 9‐LOX metabolism of anandamide (van Zadelhoff et al. Biochem. Biophys. Res. Commun. 248:33–38, 1998), we also tested this substrate with the mutants, which produced predictable shifts in product profile, including a shift from the prominent 11 S ‐hydroperoxy derivative of wild‐type to include the 15 R ‐hydroperoxide. These results conform to a model that predicts a head‐first substrate binding orientation for 9 S ‐LOX. We also found that linoleoyl‐phosphatidylcholine is not a 9 S ‐LOX substrate, which is consistent with this conclusion.