Abstract
Linoleate diol synthase (LDS) was isolated as a hemeprotein from the fungus Gaeumannomyces graminis. LDS converts linoleate sequentially to 8R-hydroperoxylinoleate (8-HPODE) through an 8-dioxygenase by insertion of molecular oxygen and to 7S,8S-dihydroxylinoleate through a hydroperoxide isomerase by intramolecular oxygen transfer. Light absorption and EPR spectra of LDS indicated that the heme iron was ferric and mainly high spin. Oxygen consumption during catalysis started after a short time lag which was reduced by 8-HPODE. Catalysis declined due to suicide inactivation. Stopped flow studies with LDS and 8-HPODE at 13 degreesC showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120 s-1. Light absorption at 406 nm then increased at a rate of approximately 4 s-1, whereas the absorption at 421 nm increased after a lag time of approximately 5 ms at a rate of approximately 70 s-1. EPR spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a transient doublet when quenched after incubation on ice for 3 s (major hyperfine splitting 2.3 millitesla; g = 2.005), indicating a protein radical. The relaxation properties of the protein radical suggested interaction with a metal center. 8-HPODE generated about twice as much radical as linoleic acid, and the 8-HPODE-induced radical appeared to be stable. Our results suggest that LDS may form, in analogy with prostaglandin H synthases, ferryl intermediates and a protein radical during catalysis.
Highlights
Presented in part at the Fourth International Congress on Essential Fatty Acids and Eicosanoids, Edinburgh, Scotland, July 20 –24, 1997, and at the 5th International Conference on Eicosanoids and Other Bioactive Lipids in Cancer, Inflammation, and Related Diseases, La Jolla, CA, September 17–20, 1997
The 13-pro-S hydrogen is abstracted by a protein radical, which is formed by oxidation of the ferriheme to ferryl oxygen intermediates during reduction of PGG2 to PGH2, or during reduction of other hydroperoxides (2, 6, 8 –10)
Our major findings are that the activity of Linoleate diol synthase (LDS) is stimulated by 8-HPODE, reduced by glutathione peroxidase, and that LDS catalysis is associated with formation of ferryl oxygen interme
Summary
Materials—Acetone powder of mycelia of G. graminis was prepared as described previously [14] and stored at Ϫ80 °C. [1-14C]18:2n-6 (55 Ci/mol) was from Amersham Pharmacia Biotech (Amersham, Buckshire, UK). Protein standards for SDS-PAGE were from Amersham Pharmacia Biotech. Lyophilized GSH peroxidase (from bovine erythrocytes; Sigma) contained 2.5% dithiothreitol, which was removed by gel filtration (PD10, Amersham Pharmacia Biotech) before use. The column was washed as described previously [14] and eluted with to 0.02 mM sodium phosphate buffer, 1 mM GSH, 0.04% Tween 20 (pH 7.4). EPR Analysis—Three different batches of purified LDS (5 M holoenzyme with a specific activity of 1–2 mol minϪ1 mgϪ1 protein) were incubated with 0.3 mM linoleic acid or with 0.1 mM 8-HPODE in 140 l for 3 or 45 s on ice and quenched (in precooled n-pentane at Ϫ105 °C). For the samples, which were incubated with 8-HPODE, the saturation curve was constructed after subtraction of a background signal from the LDS protein at the same power. After equilibration for 1–2 min, the reactions were initiated by addition of 18:2n-6 to give a concentration of 0.1 mM
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call