Description:
<jats:title>Abstract</jats:title><jats:p>Herein we characterize the <jats:italic>Arabidopsis thaliana</jats:italic> AtLOX1 and tomato (<jats:italic>Solanum lycopersicum</jats:italic>) LOXA proteins as linoleate 9<jats:italic>S</jats:italic>‐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 <jats:italic>E. coli</jats:italic> and the proteins partially purified by nickel affinity chromatography using a N‐terminal (His)<jats:sub>6</jats:sub>‐tag. Both enzymes oxygenated linoleic acid almost exclusively to the 9<jats:italic>S</jats:italic>‐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 <jats:italic>S</jats:italic>‐LOX and Gly in <jats:italic>R</jats:italic>‐LOX), revealed that substitution with Gly led to the production of a mixture of 9<jats:italic>S</jats:italic>‐ and 13<jats:italic>R</jats:italic>‐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<jats:italic>S</jats:italic>‐hydroperoxy derivative of wild‐type to include the 15<jats:italic>R</jats:italic>‐hydroperoxide. These results conform to a model that predicts a head‐first substrate binding orientation for 9<jats:italic>S</jats:italic>‐LOX. We also found that linoleoyl‐phosphatidylcholine is not a 9<jats:italic>S</jats:italic>‐LOX substrate, which is consistent with this conclusion.</jats:p>