Why do eukaryotic CE peptidases show specificity distinct from their prokaryotic counterparts? Could this indicate a profound discrepancy, hinting towards a separate origin of these two protease groups? We set out to challenge the presently held notion that ULP/ SENP proteases do not exhibit ubiquitin-specific activity [13] , while most tested bacterial homologs apparently do. To this end, we chose to biochemically define CE clan members of a more deeply branched class of eukaryotes. Pezizomycotina, a subgroup of fungi that includes A. fumigatus, A. nidulans, M. grisea, N. crassa, and G. zeae encode putative SENP8 proteases that are related in amino acid sequence to a group of yet uncharacterized bacterial C48 homologs ( Figure 1 ). In our phylogram, the common node between the respective prokaryote C48 group and SENP8 homologs of Pezizomycotina replicates with a bootstrap support of.60%. We cloned, expressed and tested the putative SENP8 protease of G. zeae, as a representative of Pezizomycotina. Unlike mammalian SENP8, the G. zeae ortholog displays dual activity towards Nedd8 and ubiquitin, similar to the previously defined CE clan protease CT868 Figure 2 . Sequence comparison between SENP8 and its homologs in human pathogenic bacteria. Multiple sequence alignment of the catalytic core region of human SENP8 (NCBI protein sequence identifier GI: 33942066, shown are residues 100-165) with the homologs in C. trachomatis (CT868, GI: 76789615, residues 199-284) [23] , E. coli (elaD, GI: 15832411, residues 228-319), L. pneumophila (GI: 52843101, residues 189-265), and S. typhi (sseL, GI: 29141091, residues 197-264) [42] . The arrows indicate active-site histidine, aspartate (or asparagine), the catalytic cysteine, and the oxyanion-stabilizing group. Predicted secondary structures are shown at the bottom and have been confirmed with the solved structure of SENP8 [27] . doi:10.1371/journal.pone.0000381.g002 Figure 3 . Biochemical assay for substrate specificity of elaD. 35 S-methionine-labeled in vitro translated wildtype elaD forms covalent adducts with suicide inhibitors based on ubiquitin (ubiquitin-vinylmethylester, VME) and Nedd8 (Nedd8-vinylsulfone, VS), but not with probes based on SUMO1 and ISG15. All probes were tested for activity with bona fide substrates (not shown) [14] . Mutation of the active-site cysteine at position 313 to serine abolishes adduct formation of elaD to electrophilic probes. Samples were resolved by reducing SDS-PAGE and visualized by fluorography. Indicated at the right is the molecular mass in kDa. doi:10.1371/journal.pone.0000381.g003 of the prokaryote C. trachomatis ( Figure 5 ) [23] . This result indicates that ubiquitin-specificity is not restricted to bacterial or viral CE peptidases, but also exists in some ancient eukaryotic members of this protease clan.