Catalytic versatility of lipoxygenase from Microcystis aeruginosa

Lipoxygenases (LOXs) catalyze the regio- and enantioselective addition of molecular oxygen to polyunsaturated fatty acids (PUFAs), yielding fatty acid hydroperoxides (FAHPs) with significant industrial relevance. Bacterial LOXs are of particular interest due to their broad substrate range and distinct regio- and enantioselectivity profiles. In the current study, we characterized the biochemical properties and product scope of a newly identified LOX from the cyanobacterium Microcystis aeruginosa (Ma-LOX). Our results demonstrate that Ma-LOX exhibits a strong preference for linoleic acid (LA) and α-linolenic acid (ALA), catalyzing regioselective dioxygen insertion predominantly at the ω-5 position for these substrates. Notably, this regioselectivity diminishes with longer-chain PUFAs. Ma-LOX shows opposite enantioselectivity with respect to eukaryotic LOXs, producing 13(R)-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE) from LA with an enantiomeric excess (ee) of 79.3 ± 8.8 % (n = 3). Structural prediction and molecular docking simulations suggest that the observed regioselectivities of Ma-LOX are influenced by oxygen insertion via two distinct pathways: a putative oxygen access channel and the entrance of a relatively shallow substrate-binding pocket, distinguishing Ma-LOX from other LOXs. Additionally, we identified that this shallow binding pocket facilitates Ma-LOX's double dioxygenation activity toward ALA, resulting in the formation of dihydroperoxides. Beyond FAHPs and dihydroperoxides, Ma-LOX catalyzes the synthesis of epoxy alcohols and ketones, suggesting the enzyme possesses an unusual but highly relevant hydroperoxide isomerase (HPI) activity. These results offer important insights into the catalytic mechanism and functional versatility of Ma-LOX, underscoring its potential for a broad range of biotechnological applications.