Abstract
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.
Highlights
Lipoxygenases (LOX) are non-heme iron dependent dioxygenases that catalyze the insertion of molecular oxygen at a (1Z,4Z)-pentadiene motif, which occurs e.g. in polyunsaturated fatty acids (PUFAs), in a regio- and stereospecific manner (An et al 2018; Liavonchanka and Feussner 2006)
Specific activities towards various PUFAs To experimentally verify the role of W330, I393 and V581 in PUFA oxygenation, we investigated the specific activities of PUFAs occurring and non-occurring in fungi of the phylum Basidiomycota
Due to the increased specific activity of the W330L and V581A variants towards the C20-22:3(ω-3)-PUFAs, the double mutant W330L/V581A was created. This variant exhibited similar or even lower specific activities towards C18-PUFA linoleic acid (C18)- as well as C20-22-PUFAs, compared to the single mutations V581A and W330L (Fig. 4a–d). Both variants V581A and V581F showed similar activities to the fungal occurring PUFAs compared to the wild type LOX, which suggests that neither increasing or decreasing space at that position leads to significantly higher activities but rather interfere with an efficient substrate binding of C18-PUFAs (Fig. 4a, b)
Summary
Lipoxygenases (LOX) are non-heme iron dependent dioxygenases that catalyze the insertion of molecular oxygen at a (1Z,4Z)-pentadiene motif, which occurs e.g. in polyunsaturated fatty acids (PUFAs), in a regio- and stereospecific manner (An et al 2018; Liavonchanka and Feussner 2006). In higher fungi of the phyla Basidiomycota, C18-PUFAs and especially linoleic acid are predominant, whereas C20-PUFAs like arachidonic acid and eicosapentaenoic acid were either found in very low amounts or in traces in fungi of the phyla Basidiomycota, such as in Cyclocybe aegerita Studies on purified basidiomycetous LOX are scarce with only LOX from Pleurotus spp. and Cyclocybe aegerita described so far (Karrer and Rühl 2019; Kuribayashi et al 2002; Leonhardt et al 2013; Plagemann et al 2013). This shortcoming is addressed in this study by site-directed mutagenesis of three amino acid residues located in the substrate tunnel of the Lox from Cyclocybe aegerita
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