Characterization of two novel fatty acid dioxygenases: Linoleate diol synthase and manganese lipoxygenase
1999 (English)Doctoral thesis, comprehensive summary (Other academic)
Two classes of fatty acid dioxygenases, PGH synthases (hemeproteins) and lipoxygenases (non-heme iron proteins), are of eminent pharmacological importance. This thesis describes a PGH synthase-related dioxygenase designated linoleate diol synthase (LDS) and the first native occurring manganese-containing lipoxygenase.
LDS was purified from the fungus Gaeumannomyces graminis as a hemeprotein with dioxygenase and hydroperoxide isomerase activities. It was previously known that the enzyme abstracts the pro-S hydrogen from the C-8 of linoleate and forms 8R-hydroperoxylinoleic acid that is sequentially isomerized to a diol, 7S,8S-dihydroxylinoleic acid. The pure enzyme was a homotetramer (520 kDa). It showed typical spectra of a high spin ferric hemeprotein, and contained at least 0.7 heme groups per monomer. A tyrosyl radical and ferryl intermediates were detected during catalysis by stopped-flow and electron paramagnetic resonance spectroscopy. With help of peptide sequence information and the reverse transcription polymerase chain reaction, the linoleate diol synthase gene was isolated from a genomic library. The gene contained three introns and spanned 3.2-kb corresponding to 978 amino acid residues. The deduced protein sequence showed homology with PGH synthases over the major catalytic domain. LDS and PGH synthases were thus found to have both structural and functional similarities.
G. graminis was found to secret a lipoxygenase. The purified enzyme contained manganese and was designated manganese-lipoxygenase (Mn-LO). Mn-LO was highly glycosylated, with a protein mass of 73 kDa. It catalyzed the conversion of linoleate into 13R-hydroperoxylinoleic acid and 11S- hydroperoxylinoleic acid, a novel lipoxygenase metabolite, by initial abstraction of the pro- S hydrogen at C-11 and insertion of O2 at C-13 or C-11 in suprafacial way. In analogy with iron-lipoxygenases, Mn-LO contained a mononuclear metal center, which was Mn(II) and oxidized during catalysis. A hypothetical lipoxygenation model for Mn-LO was presented.
In summary, LDS may belong to the same gene family as PGH synthases, the fatty acid heme dioxygenase family. The discovery of Mn-LO may have broadened our knowledge of lipoxygenases.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 1999. , 59 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 209
Pharmaceutical biosciences, Dioxygenase, hemeprotein, lipoxygenase, metalloenzyme, oxylipin, fatty acid, manganese-protein, Gaeumannomyces graminis
Research subject Pharmaceutics
IdentifiersURN: urn:nbn:se:uu:diva-358ISBN: 91-554-4520-9OAI: oai:DiVA.org:uu-358DiVA: diva2:163324
1999-10-01, lecture hall B41, Biomedical Center, Uppsala, Uppsala, 09:15