A Protein Radical and Ferryl Intermediates Are Generated by Linoleate Diol Synthase, a Ferric Hemeprotein with Dioxygenase and Hydroperoxide Isomerase Activities

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°C 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 ϳ4 s ؊1 , whereas the absorption at 421 nm increased after a lag time of ϳ5 ms at a rate of ϳ70 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.

Lipoxygenases and their metabolites in formation of plant stress tolerance

“…LOXs plants are classified considering oxidation specificity of linoleic (18 : 2) and α-linoleic (18 : 3) acids. There are iron-and manganese-compatible LOXs [12]. Mole cular weight of higher plant LOXs is within the range of 94-100 kDa [13].…”

Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation

“…Prostaglandin-endoperoxide synthase, also known as cyclooxygenase (COX), is another type of dioxygenase where a nonactivated O 2 molecule binds to a fatty acid radical generated by initial hydrogen abstraction (6,7). Whereas COX as well as linoleate diol synthase further convert the dioxygenated product with peroxidase or isomerase activity, respectively (6,8), each dioxygenase has another active site designed for the latter reaction. Contrary to the conventional one active site, one reaction paradigm, here we report that MhuD, a heme-degrading enzyme from Mycobacterium tuberculosis, catalyzes a sequential monooxygenation and dioxygenation in its single active site.…”

Chiral Phase-HPLC Separation of Hydroperoxyoctadecenoic Acids and Their Biosynthesis by Fatty Acid Dioxygenases

“…The take all fungus of wheat and several aspergilli oxidize oleic acid sequentially to (8R)-hydroperoxyoleic acid (8R-HPOME) and diols, e.g., (7S,8S)-dihydroxyoleic acid (7S,8S-DiHOME), 5S,8R-DiHOME, or 8R,11S-DiHOME (3,(5)(6)(7). The key intermediate in biosynthesis of the latter diols is 8R-HPOME, which is formed by the heme-dependent dioxygenase domains of linoleate diol synthases with both sequence and catalytic similarities to cyclooxygenases and α-dioxygenases of plants (8,9). Lipoxygenases with catalytic iron or manganese oxidize monoenoic fatty acids at low rates compared to these 8R-and 10S-dioxygenases (10).…”