Broad utilization of α-keto-β-amino acid-installing peptide chemistry in the microbial world Thomas Scott 1 , Marjan Verest 1 , Jakob Farnung 2 , Clarissa C. Forneris 1 , Serina L. Robinson 1† , Xinjian Ji 3 , Daniel U. Richter 1 , Stefanie Huber 1 , Philip Rust 1 Qi Zhang 3 , Jeffrey W. Bode 2 and Jörn Piel 1* 1 Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zürich, Switzerland, 2 Laboratory of Organic Chemistry, ETH Zürich, Switzerland, 3 Fudan University, China, † Current address: Department of Environmental Microbiology, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Switzerland Textbook knowledge of ribosomal biosynthesis is based on a conservative set of 20 proteinogenic amino acid substrates that generate products with exclusively α-amino acid-based backbone topologies. In Nature, the physiochemical limitations imposed by this restricted set of building blocks are overcome through post- translational modifications (PTMs) that substantially expand the structural and functional variation among peptides and proteins. Recently, we have uncovered a naturally occurring ‘splicease’ enzyme that catalyzes the excision of tyramine equivalents to install rare α-keto-β-amino acid moieties into ribosomal products (1). In breaking from canonical ribosomal product topology, the splicease modification achieves the installation of two highly biologically relevant chemical functionalities in a single transformation: β-Amino residues are known to confer a number of important properties including membrane permeability and proteolytic stability, and α-ketoamides are important pharmacophores in a number of natural and synthetic products with a broad spectrum of bioactivities including immunosuppression (e.g. rapamycin) and protease inhibition (e.g. Boceprevir) (2). Given the important biological activities associated with α-keto-β-amino acids, our most recent efforts have focused on expanding and exploring the splicease enzyme family. Through a systematic global bioinformatics approach and in vivo functional characterization experiments, we reveal the widespread occurrence of the splicing transformation among diverse prokaryotic phyla and a number of archaeal representatives. Splicease substrates are shown to be extremely diverse, varying in size, in amino acid composition, in the number of modification sites per substrate and in the nature of the installed β-amino residue (15 different β-residues characterized to date). We have also characterized examples of spontaneous chemistry that arise from nucleophilic α-ketoamides, further expanding the chemical diversity accessible via this PTM. Finally, we have been able to tie chemical functionality to biological activity and report exceptionally potent protease inhibitory activity for several spliced peptides from different bacterial sources, suggesting a more general role for these products in Nature. Remarkably, despite the size of this new spliced peptide (spliceotide) family, and the potent bioactivities described, there is not a single matching natural product candidate among published compounds. Our work paves the way for further investigation into the specific ecological targets of spliceotides, in addition to equipping a synthetic toolbox for the generation of novel ketoamide therapeutics. References 1. Morinaka, B.I., Lakis, E., Verest, M., Helf, M.J., Scalvenzi, T., Vagstad, A.L., Sims, J., Sunagawa, S., Gugger, M., and Piel, J. (2018). Natural noncanonical protein splicing yields products with diverse beta-amino acid residues. Science 359, 779-782. ARTN aao0157 2. 10.1126/science.aao0157.De Risi, C., Pollini, G.P., and Zanirato, V. (2016). Recent Developments in General Methodologies for the Synthesis of alpha-Ketoamides. Chemical Reviews 116, 3241-3305. 10.1021/acs.chemrev.5b00443
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