Chris Whittington, Anastasiia Kostenko, Katherine Ray, and Adam R. Offenbacher Department of Chemistry, East Carolina University, Greenville NC 27858 Lipoxygenases, including those from pathogenic fungi, catalyze the 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 across species, fungal enzymes are uniquely decorated with multiple, heterogenous N-linked glycans. The effect of these N-linked glycans on the structure and function of these enzymes remains largely unknown. One standout lipoxygenase is MoLOX, from the fungus M. oryzae, that is emerging as an important target for the devastating rice blast disease. The ability to stably label C-H bonds with deuterium enables for the assessment of rate-limiting steps of catalysis based on the magnitude of the kinetic isotope effect on the transferred hydrogen. In this presentation, we will demonstrate that hydrogen transfer, associated with C-H cleavage of the substrate linoleic acid by MoLOX, is rate determining and occurs via a hydrogen tunneling mechanism. Using the differential enthalpic barrier for hydrogen and deuterium transfer, ΔEa, 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 novel peptide:N-glycosidase. This glycosidase cleaves the N-linked carbohydrates from the protein proficiently at cool temperatures. The observed increase in ΔEa after glycosidase treatment is consistent with an impairment of both the tunneling ready state and the ground state enzyme-substrate structure. These results provide a new insight into the functional consequences of N-linked glycosylation on lipoxygenase C-H activation. Current efforts to explore the effect of other surface functionalization will be presented.

Kinetic characterization of the rate-limiting C-H activation step of M. oryzae lipoxygenase and the effects of N-linked glycosylation

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.