Clan CD forms a structural group of cysteine peptidases, containing seven families, one of which is C11 (clostripains). Information on the C11 family is limited. Here, we present the structure of a C11 peptidase, PmC11 (PDB ID: 3UWS, JCSG), from a human gut bacteria, Parabacteroides merdae. PmC11 comprises a central nine-stranded b-sheet, an unusual C-terminal extension and a break in the polypeptide chain at K147. Recombinant PmC11 has Arg/Lys specific cysteine peptidase activity, which correlates with the presence of acidic residues near the active site. Processing was found to be essential for the enzyme activity; a section of L5 (loop following β5), in a pro-form of PmC11, blocks access to the active site and the cleavage at K147 displaces the loop by ~180° allowing access to the substrates. A phylogenetic analysis of the PmC11 orthologues showed them to be present in prokaryotes, archaea, chromera, coccidia and kinetoplastida, the latter being the most divergent. We identified an orthologue in the kinetoplastid parasitic protozoan Trypanosoma brucei, TbPNT1 (Puf Nine target 1; Tb927.11.6550). Depletion of TbPNT1 in the bloodstream form by RNAi was lethal and the induced population accumulated cells lacking a kinetoplast, an organelle containing the mitochondrial DNA. We also determined that TbPNT1 is exclusively required for kinetoplast maintenance and may play a role in minicircle replication. In summary, biochemical and structural analysis of PmC11 has led us to identify an essential T.brucei protein, TbPNT1. Importantly, as there are no homologs of TbPNT1 in mammalian cells, TbPNT1 is a potential drug target. This work is supported by the MRC and JCSG is supported by NIGMS GM094586.