Distinct characteristics of the substrate binding between highly homologous catalase-related allene oxide synthase and hydroperoxide lyase

Abstract

A catalase-related allene oxide synthase (cAOS) or a hydroperoxide lyase (cHPL) fused together with an 8R-lipoxygenase is involved in the stress signaling of corals via an arachidonic acid pathway. cAOS gives rise to α-ketol and cyclopentenone, while cHPL catalyzes the cleavage of 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE) to C8-oxo acid and C12 aldehyde. In silico analysis of the substrate entry sites of highly identical coral cAOS and cHPL indicated that two positively charged residues of cAOS, K60 and K107, and the corresponding residues of cHPL, E60 and K107, may be involved in the anchoring of the carboxy group of polyunsaturated fatty acid (PUFA) hydroperoxides. A mutational analysis of cAOS and cHPL revealed that K60 or E60 and K107 were not necessary in the tethering of 8R-HpETE, however, the E60 of cHPL was essential in the productive binding of PUFA hydroperoxides. The substrate preferences of cAOS and cHPL were determined with hydroperoxy derivatives of C18, C20, C22 PUFAs, anandamide (AEA), 1-arachidonoyl glycerol (1-AG) and selected methylated substrates. Although cAOS and cHPL were able to metabolize different free PUFA substrates and arachidonoyl derivatives, only cHPL catalyzed the reaction with methylated PUFA hydroperoxides. The differences in the substrate binding and preferences between cAOS and cHPL can be explained by the distinct properties of their substrate entry sites. The current study demonstrated that homologous PUFA metabolizing enzymes may contribute to the versatile usage of the substrate pool.

Keywords: Allene oxide synthase; Catalase; Hydroperoxide lyase; Molecular modeling; Oxylipins; Site-directed mutagenesis.