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.

Radical S-adenosylmethionine (rSAM) enzymes are a large and diverse superfamily of enzymes, some of which are known to participate in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Specifically, a subfamily of rSAM proteins with an elongated C-terminus known as a SPASM domain have become a fixation in the discovery of new RiPP natural products. Arguably, a structural study, a bioinformatic study, and a functional study built the foundation of the research for rSAM-SPASM-protein-modified RiPPs. In this Review, we focus on these three studies and how they initiated what has become an increasingly productive field. In addition, we discuss the current state of RiPPs that depends on rSAM-SPASM proteins and provide guidelines to consider in future research. Lastly, we discuss how genome mining tools have become a powerful means to identify and predict new RiPP natural products. Despite the state of our current knowledge, we do not completely understand the relationship of rSAM-SPASM chemistry, substrate recognition, and the structure–function relationship as it pertains to RiPP biosynthesis, and as such, there remain many interesting findings waiting to be discovered in the future.

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Lipoxygenase (LOX) enzymes produce important cell-signaling mediators, yet attempts to capture and characterize LOX−substrate complexes by X-ray co-crystallography are commonly unsuccessful, requiring development of alternative structural methods. We previously reported the structure of the complex of soybean lipoxygenase, SLO, with substrate linoleic acid (LA), as visualized through the integration of 13 C/ 1 H electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) computations. However, this required substitution of the catalytic mononuclear, nonheme iron by the structurally faithful, yet inactive Mn 2+ ion as a spin probe. Unlike canonical Fe-LOXs from plants and animals, LOXs from pathogenic fungi contain active mononuclear Mn 2+ metallocenters. Here, we report the ground-state active-site structure of the native, fully glycosylated fungal LOX from rice blast pathogen Magnaporthe oryzae, MoLOX complexed with LA, as obtained through the 13 C/ 1 H ENDOR-guided MD approach. The catalytically important distance between the hydrogen donor, carbon-11 (C11), and the acceptor, Mn-bound oxygen, (donor−acceptor distance, DAD) for the MoLOX−LA complex derived in this fashion is 3.4 ± 0.1 Å. The difference of the MoLOX−LA DAD from that of the SLO−LA complex, 3.1 ± 0.1 Å, is functionally important, although is only 0.3 Å, despite the MoLOX complex having a Mn−C11 distance of 5.4 Å and a "carboxylate-out" substrate-binding orientation, whereas the SLO complex has a 4.9 Å Mn−C11 distance and a "carboxylate-in" substrate orientation. The results provide structural insights into reactivity differences across the LOX family, give a foundation for guiding development of MoLOX inhibitors, and highlight the robustness of the ENDOR-guided MD approach to describe LOX−substrate structures.

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 of13C/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 fromM. oryzae, MoLOX complexed with LA obtained through the13C/1H ENDOR-guided MD approach. The Mn-oxygen-to-LA donor carbon distance (DAD) forMoLOX-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 ofMoLOX inhibitors. The work highlights the robustness of ENDOR-guided MD approach to describe LOX-substrate structures that elude conventional X-ray techniques.