001 peptidyl-Asp metallopetidases is underlined; (4) the three conserved histidines (aa 167, 171, and 177), residues for zinc binding, and glutamate (aa 168), the catalytic residue, are in green; (5) two carbohydrate binding modules of the CBM_4_9 family, aa 302 to aa 432 and aa 461 to aa 586, in blue. (B) The P. aeruginosa predicted PA2783 is homologous to metalloendopeptidases from other bacteria. Interrogation of the non-redundant databases at NCBI (http://​www.​ncbi.​nlm.​nih.​gov/​; AG-881 solubility dmso accessed 10/18/2013) was done using BLASTP and the selleck inhibitor Peptidase Database MEROPS (http://​merops.​sanger.​ac.​uk/​index.​shtml; accessed 10/18/2013) was done

using BLAST. Identical aa are shown in red, similar aa in blue, and non-similar 3-Methyladenine cell line aa in black. PA2783 is homologous to the Pseudomonas mendocina ymp (Pmendo) carbohydrate-binding CenC domain-containing protein and the Ni,Fe-hydrogenase I small subunit of Hahella chejuensis KCTC 2396 (Hcheju) across the entire endopeptidase domain. Other proteins contain the HEXXHXXGXXH motif only (highlighted by a yellow box). Amacle, Alteromonas macleodii; Ahydro,

Aeromonas hydrophila; Vchole, Vibrio cholerae; Vmimic, V. mimicus; Vvulni, V. vulnificus; Xfraga, Xanthomonas fragariae; Xcampe, X. campestris; Xvesic, X. vesicatoria. Percentages of aa identity and similarity may be found in Additional file 2. PA2782 encodes a putative 22.7-kDa protein of 219 aa that contains no specific motifs, except for the presence of an alanine-rich region Pregnenolone within

its amino terminus (23 of the first 60 aa), and that has no functional homology with other known proteins (data not shown). Characterization of PA2783, a putative metalloendopeptidase The predicted protein PA2783 contains all the features of a potential endopeptidase including the putative glutamic acid catalytic residue and the three zinc-binding histidine residues within its amino terminus (Figure 5A) [39]. We tried to assess the proteolytic activity produced by PA2783 using dialyzed brain heart infusion skim milk agar. However, this approach proved unfeasible due to the production by P. aeruginosa of several proteases with strong proteolytic activities. Both PAO1/pUCP19 and PAO1/pAB2 produced identical clearing zones of protease activity (data not shown). We faced the same problem when we utilized strain PAO-R1 (Table 1), which produces a considerably reduced level of proteolytic activity due to the mutation of lasR[33]. Despite the reduction in the extracellular proteolytic activity of this strain, PAO-R1/pUCP19 and PAO-R1/pAB2 produced identical clearing zones on skim milk agar (data not shown). As an alternative, we assessed the potential proteolytic activity of PA2783 using the E. coli strain DH5α (Table 1).