title: Investigation of substrate binding and product stereochemistry issues in two linoleate 9-lipoxygenases.
abstract: 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, RRB Room 510, 23rd Ave at Pierce, Nashville, TN, 37232-6602, USA. alan.brash@vanderbilt.edu
authors.forename: William E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: Aya
authors.initials: A
authors.lastname: Itoh
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Zheng
authors.forename: Gianguido
authors.initials: G
authors.lastname: Coffa
authors.forename: Gregg A
authors.initials: GA
authors.lastname: Howe
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D019787:Linoleic Acid / Q000737:chemistry* / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018551:Solanum lycopersicum / Q000201:enzymology; D009154:Mutation; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010940:Plant Proteins; D019787:Linoleic Acid; D008084:Lipoxygenase; D000409:Alanine; D005998:Glycine
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