Description of the rate-limiting hydrogen tunneling mechanism for plant 9- and 13-lipoxygenases

Soybean lipoxygenase (GmLOX1), a plant 13-LOX, has long been considered a model protein for non-trivial, quantum hydrogen tunneling in enzyme catalysis. Hydrogen tunneling mechanisms have also been confirmed for LOXs across mammalian, fungal, and bacterial kingdoms. Notably, GmLOX1 exhibits a low activation energy (Ea) for catalysis relative to characterized non-plant LOXs. In the present study, we examined three additional plant isozymes, including representatives of both 9- and 13-LOXs. All plant LOXs show large primary deuterium kinetic isotope effects associated with rate-limiting hydrogen tunneling during cleavage of a carbon-hydrogen bond on substrate linoleic acid. The monomeric plant 9-LOXs studied herein uniquely display kinetic cooperativity in their steady-state kinetics, while GmLOX1 does not. These data implicate fatty acid-dependent structural regulation of plant 9-LOX catalysis in a similar manner to mammalian LOXs. In lieu of experimental structures of these plant LOXs with substrate, we predicted representative 9- and 13-LOX isoforms in AlphaFold3 (AF3) with fatty acids and compared them with their crystal structures to better predict modes of substrate acquisition that could support the alternative kinetic models. Further, we find that two 9-LOXs have larger Ea values relative to GmLOX1. To understand this property, we also present a visualization of LOX evolutionary conservation across the plant kingdom. The data provide deeper insights into the reaction mechanisms of plant LOXs in the context of the activation barriers for catalysis and the relationships to the thermal activation of hydrogen tunneling.