Leucine/valine residues direct oxygenation of linoleic acid by (10R)- and (8R)-dioxygenases: expression and site-directed mutagenesis oF (10R)-dioxygenase with epoxyalcohol synthase activity

J Biol Chem. 2009 May 15;284(20):13755-13765. doi: 10.1074/jbc.M808665200. Epub 2009 Mar 16.

Abstract

Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; approximately 90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; approximately 10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms approximately 98% 8R-HPODE and approximately 2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aspergillus fumigatus / enzymology*
  • Aspergillus fumigatus / genetics
  • Cell Line
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Gene Expression*
  • Insecta
  • Intramolecular Oxidoreductases / biosynthesis*
  • Intramolecular Oxidoreductases / chemistry*
  • Intramolecular Oxidoreductases / genetics
  • Intramolecular Oxidoreductases / isolation & purification
  • Leucine / chemistry
  • Leucine / genetics
  • Leucine / metabolism
  • Ligases / biosynthesis*
  • Ligases / chemistry*
  • Ligases / genetics
  • Ligases / isolation & purification
  • Linoleic Acid / chemistry
  • Linoleic Acid / genetics
  • Linoleic Acid / metabolism
  • Linoleic Acids / chemistry
  • Linoleic Acids / genetics
  • Linoleic Acids / metabolism
  • Mutagenesis, Site-Directed
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Valine / chemistry*
  • Valine / genetics
  • Valine / metabolism

Substances

  • Fungal Proteins
  • Linoleic Acids
  • Recombinant Proteins
  • 8-hydroperoxylinoleic acid
  • Linoleic Acid
  • Intramolecular Oxidoreductases
  • hydroperoxide isomerase
  • Ligases
  • Leucine
  • Valine