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Investigation of substrate binding and product stereochemistry issues in two linoleate 9-lipoxygenases.

Boeglin WE, Itoh A, Zheng Y, Coffa G, Howe GA, Brash AR
Lipids. 2008 43 (11): 979-87

PMID: 18795358 · PMCID: PMC4710486 · DOI:10.1007/s11745-008-3230-1

Herein we characterize the Arabidopsis thaliana AtLOX1 and tomato (Solanum lycopersicum) LOXA proteins as linoleate 9S-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 9S-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 9S- and 13R-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 11S-hydroperoxy derivative of wild-type to include the 15R-hydroperoxide. These results conform to a model that predicts a head-first substrate binding orientation for 9S-LOX. We also found that linoleoyl-phosphatidylcholine is not a 9S-LOX substrate, which is consistent with this conclusion.

MeSH Terms (15)

Alanine Arabidopsis Binding Sites Catalysis Catalytic Domain Chromatography, High Pressure Liquid Glycine Linoleic Acid Lipoxygenase Lycopersicon esculentum Mutation Phylogeny Plant Proteins Stereoisomerism Substrate Specificity

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