PqqL is an iron regulated, periplasmically localized, metallopeptidase, with an elongated conformation.

A) An anti-PqqL western blot of E. coli BW25113 or CFT073 whole cells grown in the presence or absence of the iron chelator 2’2-bipyridine (BP) or in human urine. Detection of SurA is shown as a loading control. Left shows a representative blot, right shows normalised intensity of blots from 3 biological replicates. This shows more PqqL is produced under iron-limiting conditions. Indicated significant differences between conditions are based on student’s t-test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). B) A western blot of cell fractions from E. coli BW25113 grown under iron-limiting conditions (100 μM BP), showing the distribution of PqqL in whole cell (WC), outer envelope (OE) and periplasmic (PP) fractions, but not associated with the outer membrane (OM). Controls using antisera recognizing SurA (periplasmic), BamA (outer membrane) or YtfP (cytoplasmic) are shown. Left shows a representative blot, right shows quantified intensity of this blot C) The crystal structure of PqqL showing that its two ‘clam shell’ domains adopt a highly elongated conformation in the crystal structure, engaging in minimal intra-molecular contacts. D) The crystal structure of PqqL illustrates the presence of a putative Zn ion in the protease active site of the enzyme. E) Peptidase screening assays shows that PqqL is an active peptidase with specificity for peptides containing a F/Y, F/Y, V/A motif (Full data shown in S4 Table).