A bisallylic mini-lipoxygenase from cyanobacterium Cyanothece sp. that has an iron as cofactor.
Lipoxygenases are enzymes that are found ubiquitously in higher animals and plants, but have only recently been identified in a number of bacteria. The genome of the diazotrophic unicellular cyanobacterium Cyanothece sp. harbors two genes with homology to lipoxygenases. Here we describe the isolation of one gene, formerly named csplox2. It was cloned, and the protein was expressed in Escherichia coli and purified. The purified enzyme belongs to the group of prokaryotic mini lipoxygenases, because it had a molecular mass of 65 kDa. Interestingly, it catalyzed the conversion of linoleic acid, the only endogenously found polyunsaturated fatty acid, primarily to the bisallylic hydroperoxide 11R-hydroperoxyoctadecadienoic acid. This product had previously only been described for the manganese lipoxygenase from the take all fungus, Gaeumannomyces graminis. By contrast, CspLOX2 was shown to be an iron lipoxygenase. In addition, CspLOX2 formed a mixture of typical conjugated lipoxygenase products, e.g. 9R- and 13S-hydroperoxide. The conversion of linoleic acid took place with a maximum reaction rate of 31 s(-1). Incubation of the enzyme with [(11S)-(2)H]linoleic acid led to the formation of hydroperoxides that had lost the deuterium label, thus suggesting that CspLOX2 catalyzes antarafacial oxygenation as opposed to the mechanism of manganese lipoxygenase. CspLOX2 could also oxidize diarachidonylglycerophosphatidylcholine with similar specificity as the free fatty acid, indicating that binding of the substrate takes place with a "tail-first" orientation. We conclude that CspLOX2 is a novel iron mini-lipoxygenase that catalyzes the formation of bisallylic hydroperoxide as the major product.
Andreou
Alexandra
A
Department of Plant Biochemistry, Albrecht von Haller Institute for Plant Sciences, Georg August University, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Göbel
Cornelia
C
Hamberg
Mats
M
Feussner
Ivo
I
D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008084:Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism
D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D007501:Iron; D008084:Lipoxygenase