Fig 1. Metal dependence of PprI protease.

(A) PprI cleavage activity in vitro was measured using purified PprI and DdrO protein. PprI cleaves DdrO into two fragments as indicated by the SDS-PAGE gel. (B) Metal ion scan for the protease activity. From left to right: without additive, 1 mM EDTA, 1 mM CaCl₂, 1 mM FeCl₂, 1 mM MgCl₂, 1 mM NiSO₄, 1 mM CuCl₂, 1 mM MnCl₂, 1 mM ZnCl₂. Mn(2+) is necessary for the protease activity. (C) Mutations at the metal-binding sites of PprI abolish the proteolytic activity. From left to right: wild type PprI, H118L mutant, E119Q mutant, H122L mutant and E149Q mutant. These mutations were chosen based on the crystal structure from D. deserti (PDB code 3DTI) [9].