Investigation of Substrate Binding and Product Stereochemistry Issues in Two Linoleate 9-lipoxygenases

Lipids. 2008 Nov;43(11):979-87. doi: 10.1007/s11745-008-3230-1. Epub 2008 Sep 16.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alanine / genetics
  • Alanine / metabolism
  • Arabidopsis / enzymology
  • Binding Sites
  • Catalysis
  • Catalytic Domain
  • Chromatography, High Pressure Liquid
  • Glycine / genetics
  • Glycine / metabolism
  • Linoleic Acid / chemistry*
  • Linoleic Acid / metabolism
  • Lipoxygenase / chemistry*
  • Lipoxygenase / genetics
  • Lipoxygenase / isolation & purification
  • Lycopersicon esculentum / enzymology
  • Mutation
  • Phylogeny
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification
  • Stereoisomerism
  • Substrate Specificity

Substances

  • Plant Proteins
  • Linoleic Acid
  • Lipoxygenase
  • Alanine
  • Glycine