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© The Author(s) 2020. ; Flagellins are the protein components of bacterial flagella and assemble in up to 20,000 copies to form extracellular flagellar filaments. An unusual family of flagellins was recently discovered that contains a unique metalloprotease domain within its surface-exposed hypervariable region. To date, these proteolytic flagellins (also termed flagellinolysins) have only been characterized in the Gram-positive organism Clostridium haemolyticum, where flagellinolysin was shown to be proteolytically active and capable of cleaving extracellular protein substrates. The biological function of flagellinolysin and its activity in other organisms, however, remain unclear. Here, using molecular biochemistry and proteomics, we have performed an initial characterization of a novel flagellinolysin identified from Hylemonella gracilis, a Gram-negative organism originally isolated from pond water. We demonstrate that H. gracilis flagellinolysin (HgrFlaMP) is an active calcium-dependent zinc metallopeptidase and characterize its cleavage specificity profile using both trypsin and GluC-derived peptide libraries and protein substrates. Based on high-throughput degradomic assays, HgrFlaMP cleaved 784 unique peptides and displayed a cleavage site specificity similar to flagellinolysin from C. haemolyticum. Additionally, by using a set of six protein substrates, we identified 206 protein-embedded cleavage sites, further refining the substrate preference of HgrFlaMP, which is dominated by large hydrophobic amino acids in P1′, and small hydrophobic or medium-sized polar residues on the amino-terminal side of the scissile bond. Intriguingly, recombinant HgrFlaMP was also capable of cleaving full-length flagellins from another species, suggesting its potential involvement in interbacterial interactions. Our study reports the first experimentally characterized proteolytic flagellin in a Gram-negative organism, and provides new insights into flagellum-mediated enzymatic activity. ; This study was supported by a post-doctoral fellowship from the Peter and Traudl Engelhorn Foundation (U.E.) and the Austrian Science Fund (FWF, project number: W1213; H.B., C.B. and C.G.H.). A.C.D. is supported by an Ontario Early Research Award and by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Discovery Grant. U.E. is currently supported by the Beatriu de Pinós Program (2020-2023), funded by the Secretary of Universities and Research (Government of Catalonia) and by the Horizon 2020 program of research and innovation of the European Union under the Marie Sklodowska-Curie COFUND actions. Michael Mansfeld gratefully acknowledges funding from the Japan Society for the Promotion of Science as a JSPS International Research Fellow (Luscombe Unit, Okinawa Institute of Science and Technology Graduate University), and Michael Kohlberger is acknowledged for technical assistance during PICS library preparations. Financial support for the Christian Doppler Laboratory for Biosimilar Characterization by the Austrian Federal Ministry of Science, Research, and Economy and by a Start-up Grant of the State of Salzburg is gratefully acknowledged.