Lee, H. G. | bioRxiv Science

bioRxiv2021

Molecular mechanism underlying substrate recognition of the peptide macrocyclase PsnB

Graspetides, also known as omega-ester-containing peptides (OEPs), are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) bearing side-to-side macrolactone or macrolactam linkages. Here we present molecular details of the precursor recognition of the macrocyclase enzyme PsnB in the biosynthesis of plesiocin, a Group 2 graspetide. Biochemical analysis revealed that, in contrast to other RiPPs, the core region of the plesiocin precursor peptide noticeably enhanced the enzyme-precursor interaction via the conserved glutamates. We obtained four crystal structures of symmetric or asymmetric PsnB dimers including those with a bound core peptide and a nucleotide, and suggest that the highly conserved Arg213 at the enzyme active site specifically recognizes a ring-forming acidic residue and escorts it to ATP for phosphorylation. Collectively, this study provides insights into the mechanism underlying substrate recognition in the graspetide biosynthesis, and lays a foundation for engineering new variants.

biochemistry↗

bioRxiv2020

MAP Kinase OsMEK2 and OsMPK1 Signaling for Ferroptotic Cell Death in Rice-Magnaporthe oryzae Interactions

ABSTRACTMitogen-activated protein kinase (MAPK) signaling is required for plant cell death responses to invading microbial pathogens. Ferric ions and reactive oxygen species (ROS) accumulate in rice (Oryza sativa) tissues undergoing cell death during Magnaporthe oryzae infection. Here, we report that rice MAP kinase (OsMEK2 and OsMPK1) signaling cascades are involved in iron- and ROS-dependent ferroptotic cell death responses of rice to M. oryzae infection. OsMEK2 interacted with OsMPK1 in the cytoplasm, and OsMPK1 moved from the cytoplasm into the nucleus to bind to the OsWRKY90 transcription factor. OsMEK2 expression may trigger OsMPK1-OsWRKY90 signaling pathways in the nucleus. Avirulent M. oryzae infection in ΔOsmek2 mutant rice did not trigger iron and ROS accumulation and lipid peroxidation, and also downregulated OsMPK1, OsWRKY90, OsRbohB, and OsPR-1b expression. However, OsMEK2 overexpression induced ROS-and iron-dependent cell death in rice during M. oryzae infection. The downstream MAP kinase (OsMPK1) overexpression induced ROS- and iron-dependent ferroptotic cell death in the compatible rice-M. oryzae interaction. These data suggest that the OsMEK2-OsMPK1-OsWRKY90 signaling cascade is involved in the ferroptotic cell death in rice. The small-molecule inducer erastin triggered iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death in ΔOsmek2 mutant plants during M. oryzae infection. Disease-related cell death was lipid ROS-dependent and iron-independent in the ΔOsmek2 mutant plants. These combined results suggest that OsMEK2 and OsMPK1 expression positively regulates iron- and ROS-dependent ferroptotic cell death via OsMEK2-OsMPK1-OsWRKY90 signaling pathways, and blast disease (susceptibility)-related cell death was ROS-dependent but iron-independent in rice-M. oryzae interactions.View Full Text

molecular biology↗