<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-6625 |
| Date | January 2006 |
| Creators | Cristea, Mirela |
| Publisher | Uppsala University, Department of Pharmaceutical Biosciences, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, 1651-6192 ; 28 |
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