Kinetic Characterization of the C–H Activation Step for the Lipoxygenase from the Pathogenic Fungus Magnaporthe oryzae: Impact of N‑Linked Glycosylation

Lipoxygenases from pathogenic fungi belong to the lipoxygenase family of enzymes, which catalyze C–H activation of polyunsaturated fatty acids to form a diverse set of cell-signaling hydroperoxides. While the lipoxygenase catalytic domains are structurally and functionally similar, these fungal enzymes are decorated with N-linked glycans. The impact of N-linked glycans on the structure and function of these enzymes remains largely unknown. One exemplary system is MoLOX, a lipoxygenase from the fungus Magnaporthe oryzae, that is emerging as an important target for the devastating rice blast disease. Herein, we demonstrate that hydrogen transfer, associated with C–H cleavage of the substrate linoleic acid by MoLOX, is rate-determining and occurs by a hydrogen tunneling mechanism. Using the differential enthalpic barrier for hydrogen and deuterium transfer, ΔE a, as a kinetic reporter of tunneling efficiency, a disproportionate increase in the activation energy for deuterium transfer is observed upon treatment of MoLOX with a peptide:N-glycosidase that cleaves N-linked carbohydrates from the protein. This increased ΔE a is consistent with an impairment of substrate positioning in the enzyme–substrate complex for both the tunneling ready state and the ground state. These results provide new insight into the functional consequences of N-linked glycosylation on lipoxygenase C–H activation and have important implications for MoLOX inhibitor design.