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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.

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.

SUBMITTER: Garscha U

PROVIDER: S-EPMC2679477 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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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.

Garscha Ulrike U Oliw Ernst H EH

The Journal of biological chemistry 20090316 20

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-LD ...[more]

PMID: 19289462

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Project description:Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).

Dataset Information

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.

Publications

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.

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