Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein.
The fungus Gaeumannomyces graminis, which causes the major root disease of wheat known as "take-all," can metabolize linoleic acid to (8R)-hydroperoxylinoleic acid. The enzyme linoleate 8-dioxygenase abstracts hydrogen and introduces molecular oxygen in an antarafacial way at C-8. We have now purified the enzyme 1000-fold to a specific activity of 1.8 micronol/min/mg of protein. Acetone powder of mycelia of G. graminis was subjected to extraction and ammonium sulfate precipitation with solubilization. The 8-dioxygenase was purified by hydrophobic interaction chromatography, size-exclusion chromatography, anion-exchange chromatography, and immobilized metal ion affinity chromatography. The active enzyme appeared to consist of four subunits since the active enzyme had an apparent molecular mass of 520 kDa determined by gel filtration, while SDS-polyacrylamide gel electrophoresis showed a protein band of 130 kDa. Spectroscopy indicated the presence of heme. The characteristic pyridine ferrohemochrome alpha-band was found at 557 nm and the beta-band at 525 nm. The purified protein showed an absorption maximum at 408 nm (gamma, Soret). The absorption maximum shifted to 429 nm after reduction with dithionite and to 421 nm after treatment of the reduced enzyme with carbon monoxide. BW A4C, a hydroxamic acid derivative, inhibited the enzyme by >90% at 10 microM. The pH optimum was 7.2-7.4, the isoelectric point was 5.2 by chromatofocusing, and the Km values were 8 microM for linoleic acid and 30 microM for oxygen. We conclude that linoleate 8-dioxygenase appears to be a tetrameric hemoprotein distinct from other fatty-acid dioxygenases.
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Department of Pharmaceutical Biosciences, Uppsala University Biomedical Center, Box 591, S-751 24 Uppsala, Sweden.
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D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006420:Hemeproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013053:Spectrophotometry; D013098:Xylariales / Q000201:enzymology*
D006420:Hemeproteins; D046911:Macromolecular Substances; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase