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Evidence for the mitochondrial biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid in the yeast Dipodascopsis uninucleata

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Lipids

Abstract

The biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE) from arachidonic acid (20∶4n−6) by the hyphal-forming yeast, Dipodascopsis uninucleata, in cell-free enzyme extracts required CoASH, ATP, NAD+ and Mg2+; 3R-HETE was present as the CoA derivative in enzyme extracts and its biosynthesis was associated with mitochondria. Its synthesis was high from arachidonoyl-CoA (15% conversion of the substrate; 22 nmol mg protein−1·h), but significantly higher from trans-2-arachidonoyl-CoA (53 nmol mg protein−1·min). Aspirin, an inhibitor of prostaglandin endoperoxide synthase synthase (cyclooxygenase), did not significantly inhibit 3R-HETE biosynthesis in enzyme extracts, as opposed to antimycin A (46% inhibition). The chirality of 3-HETE was 95% R and 5% S. 3R-HETE has the same chirality as the products of peroxisomal enoyl-CoA hydratases of Neurospora crassa and Saccharomyces cerevisiae; the difference appears to be that in D. uninucleata the R-enantiomers are synthesized in mitochondria. Exogenously supplied eicosapentaenoic acid was converted to 3-hydroxy 5Z,11Z,14Z,17Z-eicosapentaenoic acid by cell-free enzyme extracts though there was no requirement for a 5Z,8Z-diene structure for the biosynthesis of 3-hydroxylated fatty acids as 3-hydroxy-8Z,11Z,14Z, and 3-hydroxy-11Z,14Z,17Z-eicosatrienoic acids were synthesized from the corresponding fatty acids. We found no evidence for the synthesis of the prostaglandins F2α and F2.

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Abbreviations

20∶3n−6:

8Z,11Z,14Z-eicosatrienoic acid

20∶3n−3:

11Z,14Z,17Z-eicosatrienoic acid

20∶4n−6:

5Z,8Z,11Z,14Z-eicosatetraenoic acid

20∶5n−3:

5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid

20∶4n−6-CoA:

5Z,8Z,11Z,14Z-eicosatetraenoyl-CoA

BSA:

bovine serum albumin

EI:

electron impact

EM:

electron microscopy

GC:

gas chromatography

3R-HETE:

3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid

3R-HETE-CoA:

3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoyl-CoA

HPLC:

high-pressure liquid chromatography

int. diam.:

internal diameter

MS:

mass spectrometry

NDGA:

nordihydroguaiaretic acid

NS-398:

N-(2cyclohexyloxy-4-nitrophenyl)-methane sulfonamide

PGF2α :

prostaglandin F2α

PGE2 :

prostaglandin E2

PGH synthase:

prostaglandin endoperoxide synthase

RCR:

respiratory control ratio

RP:

reversed-phase

sp. act.:

specific activity

TLC:

thin-layer chromatography

Tris/HCl:

Tris (hydroxymethyl) methylamine hydrochloride

YNB:

yeast nitrogen base

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Authors and Affiliations

  1. Department of Biological Sciences, University of Hull, HU6 7RX, Hull, UK

    Simon R. Fox, John Friend & Colin Ratledge

  2. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden

    Mats Hamberg

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  1. Simon R. Fox
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  2. Mats Hamberg
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  3. John Friend
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  4. Colin Ratledge
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Correspondence to Simon R. Fox.

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Fox, S.R., Hamberg, M., Friend, J. et al. Evidence for the mitochondrial biosynthesis of 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid in the yeast Dipodascopsis uninucleata . Lipids 35, 1205–1214 (2000). https://doi.org/10.1007/s11745-000-0637-4

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  • DOI: https://doi.org/10.1007/s11745-000-0637-4

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