Abstract
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.
Highlights
Oxylipins are lipoxygenase-derived metabolites with a variety of different structures and biological functions
LOX separated into two distinct sections with the characterized LOX from G. graminis [19] on one side and all basidiomycetous LOX as well as the characterized LOX from F. oxysporum [20] on the other side (S1 Fig)
The observed influence of temperature and pH on AaeLOX4 with its maximum activity at 25 ̊C and pH 7.5 is in accordance with LOXs from other fungi, such as a wildtype LOX from P. ostreatus, having its maximum activity at 25 ̊C and pH values between 7.5 and 8.0 [10]
Summary
Oxylipins are lipoxygenase-derived metabolites with a variety of different structures and biological functions. Human lipid mediators like e.g. hepoxilins and trioxilins, which play an important role in tissue healing, organ protection, pain reduction and host defense received a lot of attention [2, 3]. In plants, they serve as signaling molecules, are involved in mediate stress response or in defense against pathogens, whereas in fungi they are associated with sporulation and the sexual as well as asexual life cycle [4, 5]. Lipoxygenases (LOX) are non-heme iron dependent dioxygenases which catalyze the insertion of molecular oxygen at a (1Z,4Z)-pentadiene motif, which
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