Structure function studies of homologous plant lipoxygenase

Abstract

Enzymes are the catalysts of biological reactions that lower the barrier of activation energy needed for the reaction to occur. The lipoxygenase, LOX, family of enzymes is found in all kingdoms and catalyzes the oxidation of polyunsaturated fatty acids. LOXs play diverse roles in plants by affecting growth factors and pathogenic defense while human LOXs contribute to multiple inflammation pathways related to health and disease. The plant isozyme, soybean lipoxygenase (SLO), has long served as a robust model for mechanistic studies of the LOX reaction. The LOX reaction is initiated by an irreversible C-H cleavage by the LOX's non-heme, mononuclear iron cofactor. Large, temperature independent kinetic isotope effects support a tunneling mechanism for H transfer. The SLO reaction with LA is also associated with a low activation energy (Ea) whereas fungal, animal, and bacterial enzymes have displayed larger Ea values. Studies have shown a solvent-exposed loop in SLO linked to the network of thermal energy that mediates the tunneling mechanism. This same exposed loop and catalytic efficiency is not retained in mammalian LOX and is not well studied in plants other than SLO. In this thesis, a library of homologous plant LOXs is sequenced, isolated from recombinant expressions in bacteria cultures, kinetically examined, and structurally studied in reference to SLO, the model protein. This aims to examine the prevalence of efficient catalysis in homologous plant LOX while also identifying structural and sequential similarities conserved with SLO.