Bacterial cyclooxygenase homologs might be associated with multicellularity Georgy Kurakin1 and Nadezhda Potapova2 1Pirogov Russian National Research Medical University, Moscow, Russia, email: georgykurakin@gmail.com ❑Lipoxygenases (LOXs) could be evolutionarily associated with multicellularity (Paper 1, Paper 2). ❑Some bacteria might use LOXs for cross-kingdom pathogenicity and immune response evasion. ❑Oxylipins are almost universal signalling compounds across all multicellular eukaryotes. ❑Oxylipin biosynthesis is provided by homologous lipoxygenase- like and cyclooxygenase-like enzymes in different kingdoms. What did we know? What was recently found? What about cyclooxygenase-like (COX-like) enzymes? Are they associated with multicellularity? NO- 2 ❑We constructed a series of phylogenetic trees of bacterial cyclooxygenase homologs, animal cyclooxygenases (COXs), plant α-dioxygenases (αDOXs), and fungal COX homologs. ❑Some of these enzymes perform different function, but a biochemically characterized Nostoc COX-like enzyme with a lipoxygenase activity (in the green cluster) indicated a supercluster of bacterial enzymes with probable oxylipin biosynthesis finction. We called it “Our cluster”. 2Kharkevich Institute of Information Transmission Problems RAS, Moscow, Russia, email: nadezhdalpotapova@gmail.com LOX Alginate lyase Pseudomonas aeruginosa LOX LOX LOX Cyp 450 FA desaturase Myxobacteria COX Nitrosomonadales Cyanobacteria Cyp 450 24 15 68 Confidences for Nostoc punctiforme Confidences for Myxococcus xanthus 36 Confidences for Nitrosomonas europaea 1 Confidences for Pseudomonas aeruginosa Indirect extra-phylogenetic confirmation of the close proximity of animal COX and nitrosomonadal homologs: LOX and COX-like protein in one operon in Nitrosomonadales and their functional synergy in animals. (OperonDB data). COX1 ovine ppoC Asp. Fum. αDOX COX-like Nostoc Myeloperoxidase V116 R V L R120 L L L F205 I M F F209 I W F Y348 Y W Y V349 L T I L352 I L I Y355 L T Y N375 N Y N I377 C L M F381 F F F F Y385 Y Y Y F W387 W M W Y M522 I A V I523 A F A G526 Y F I A527 T L G S530 A A A G533 L R Q L534 V L A E-val ~10-11 ~10-4 N.S.S. Substrate-binding residues are conserved between COXs ans Nostoc COX-like protein, but evolved independently in αDOXs and fungal COX-like enzymes. COX PDB ID: 1diy This is confirmed by the substrate placement in COX and αDOX — it is almost opposite (PDB IDs: 1diy, 4kvl, UCSF Chimera model). αDOX PDB ID: 4kvl COX PDB ID: 1diy Nostoc enzyme Model, docking In contrast, the docking results suggest that the bioclemically characterized Nostoc enzyme might bind the substrate like COX (AutoDock Vina docking, UCSF Chimera model). conserved carboxylate- binding Tyr Corynebacteriales Burkholderiales Caulobacterales Rhizobiales Rhodobacterales Oceanospirillales Nitrosomonadales Sphingomonadales Rhodospirillales Geodermatophilales Propionibacteriales Streptosporangiales Methylococcales Alteromonadales Synechococcales Nostocales Chroococcales Oscillatoriales Streptomycetales 0 2 4 6 8 10 12 COX-like enzyme occurrence ❑We have obtained the indirect evidence that “our cluster” proteins are oxylipin-producing enzymes of bacteria closely related to our COXs. ❑The occurrence of “our cluster” enzymes (at the left) is at the highest in filamentous cyanobacteria and Streptosporangiales actinobacteria. They are multicellular. ❑This leads to a conclusion that COX-like enzymes in bacteria are statistically associated with multicellularity (like LOXs). ❑They might be involved in oxylipin cell-to-cell signalling for maintaining complex structure, like human COXs and plant αDOXs. Find out more in our slide deck on ResearchGate: Kurakin, G.F., Potapova, N.A. (2023) Insights into the cyclooxygenase phylogeny. Unpublished. https://doi.org/10.13140/RG.2.2.35390.46402 Methods and full references are included. ResearchGate (@ResearchGate) | Twitter BioRender - Crunchbase Company Profile Funding Created with BioRender.com We used Tree visuali