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A single mutation (Gly316Ala) of manganese lipoxygenase augments the hydroperoxide isomerase activity.: Mechanism of biosynthesis of epoxyalcohols
Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Article in journal (Refereed) Submitted
URN: urn:nbn:se:uu:diva-94111OAI: oai:DiVA.org:uu-94111DiVA: diva2:167855
Available from: 2006-03-22 Created: 2006-03-22Bibliographically approved
In thesis
1. Expression of Manganese Lipoxygenase and Site-Directed Mutagenesis of Catalytically Important Amino Acids: Studies on Fatty Acid Dioxygenases
Open this publication in new window or tab >>Expression of Manganese Lipoxygenase and Site-Directed Mutagenesis of Catalytically Important Amino Acids: Studies on Fatty Acid Dioxygenases
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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).

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 Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.

Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris 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.

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”).

Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.

The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis.

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 60 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 28
Pharmaceutical pharmacology, Dioxygenase, Gaeumannomyces graminis, lipoxygenase, Magnaporthe grisea, oxylipin, Pichia pastoris, hydroperoxide isomerase, polyunsaturated fatty acids, Farmaceutisk farmakologi
National Category
Pharmacology and Toxicology
urn:nbn:se:uu:diva-6625 (URN)91-554-6487-4 (ISBN)
Public defence
2006-04-07, B41, Biomedical Centre, Box 591, BMC, Uppsala, 09:15
Available from: 2006-03-22 Created: 2006-03-22Bibliographically approved

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