13C ENDOR Spectroscopy-Guided MD Computations Reveals the Structure of the Enzyme-Substrate Complex of an Active, N-linked Glycosylated Lipoxygenase

Lipoxygenases (LOXs) are enzymes responsible for producing important cell signaling mediators and have been extensively studied for their potential clinical relevance as well as to advance our understanding of enzyme catalysis. The common inability to capture and characterize LOX-substrate complexes by Xray co-crystallography requires the development of alternative structural methods. We previously reported the integration of 13C/1H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) to visualize the complex structure of the paradigmatic LOX from soybean, SLO, with substrate linoleic acid (LA). However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn2+ ion as a spin-probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear manganese metallocentres. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from M. oryzae, MoLOX complexed with LA obtained through the 13C/1H ENDOR-guided MD approach. The Mn-oxygen-to-LA donor carbon distance (DAD) for MoLOX-LA, 3.4 ± 0.3 Å, matches the distance in the single representative X-ray co-structure of an animal 8R-LOX with its natural fatty acid substrate, and slightly elongated from that of the SLO-LA complex, 3.1 ± 0.2 Å, despite its ‘carboxylate-out’ substrate binding orientation versus ‘carboxylate-in’ for SLO. The results provide unique insight into the evolutionary divergence of the ground-state DAD in the LOX family, which influences the activation barrier for hydrogen tunneling, and give a structural basis for guiding development of MoLOX inhibitors. The work highlights the robustness of ENDOR-guided MD approach to describe LOX-substrate structures that elude conventional X-ray techniques.