Lipoxygenases (LOXs), 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 LOX catalytic domains are structurally and functionally similar across species, fungal enzymes are uniquely decorated with multiple, heterogenous N-linked glycans. N-linked glycans are considered to play an important role in protein stability and solubility as well as cell signaling and glycoprotein trafficking. Removal of these post translational modifications are often necessary for structural characterization for structure-function relationships and rational inhibitor design. The common methods include mutagenesis (e.g. AsnàGln) or enzymatic treatment (EndoH or PNGaseF). How these enzymes influence the structure and proficiency of enzymes is not well understood. A model fungal lipoxygenase is MoLOX, from the fungus M. oryzae, that is emerging as an important target for the devastating rice blast disease. MoLOX has eight predicted sites of N-linked glycans that have been confirmed by mass spectrometry and site-directed mutagenesis. While a high-resolution X-ray structure of MoLOX, treated by EndoH, has been solved, the influence of EndoH processing on the impact of the MoLOX active site structure is unresolved. In this presentation, a combination of kinetic isotope effects, substrate selectivity analysis, hydrogen deuterium exchange-mass spectrometry (HDX-MS), and electron-nuclear double resonance (ENDOR) spectroscopy is presented to assess the structural and functional differences among four protein variants – the native enzyme, EndoH-treated and PNGaseF-treated enzymes, and a AsnàGln octomutant of MoLOX. The latter harbors no N-linked glycans. These results shed new insights into the structural and functional consequences of N-linked glycosylation changes on lipoxygenase C-H activation.

Kinetic and biophysical characterization of the impact of N-linked glycosylation on protein-ligand structure and proficiency of enzymatic C-H activation in a fungal lipoxygenase

ACS Spring Meeting 2021

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