RESUMO
The basidiomycetous lipoxygenase Lox1 from Cyclocybe aegerita catalyzes the oxygenation of polyunsaturated fatty acids (PUFAs) with a high preference towards the C18-PUFA linoleic acid (C18:2 (ω-6)). In contrast, longer PUFAs, generally not present in the fungal cell such as eicosatrienoic acid (C20:3(ω-3)) and docosatrienoic acid (C22:3 (ω-3)), are converted with drastically lower activities. With site-directed mutagenesis, we were able to create two variants with enhanced activities towards longer chain PUFAs. The W330L variant showed a ~ 20 % increased specific activity towards C20:3(ω-3), while a ~ 2.5-fold increased activity against C22:3 (ω-3) was accomplished by the V581 variant.
RESUMO
Oxylipins are metabolites with a variety of biological functions. However, the biosynthetic pathway is widely unknown. It is considered that the first step is the oxygenation of polyunsaturated fatty acids like linoleic acid. Therefore, a lipoxygenase (LOX) from the edible basidiomycete Agrocybe aegerita was investigated. The AaeLOX4 was heterologously expressed in E. coli and purified via affinity chromatography and gel filtration. Biochemical properties and kinetic parameters of the purified AaeLOX4 were determined with linoleic acid and linolenic acid as substrates. The obtained Km, vmax and kcat values for linoleic acid were 295.5 µM, 16.5 µM · min-1 · mg-1 and 103.9 s-1, respectively. For linolenic acid Km, vmax and kcat values of 634.2 µM, 19.5 µM · min-1 · mg-1 and 18.3 s-1 were calculated. Maximum activities were observed at pH 7.5 and 25 °C. The main product of linoleic acid conversion was identified with normal-phase HPLC. This analysis revealed an explicit production of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD). The experimental regio specificity is underpinned by the amino acid residues W384, F450, R594 and V635 considered relevant for regio specificity in LOX. In conclusion, HPLC-analysis and alignments revealed that AaeLOX4 is a 13-LOX.
