Figure 6

Analysis of critical residues in the fingerprint region of YgfZ. (A) Inhibition zone assay for the plumbagin-countering activity of amino acid-substituted YgfZ. The ΔygfZ mutant was transformed with pQE-ygfZ-derived plasmids to express variants of E. coli YgfZ. K226A, G227A, C228A, Y229A, T230A, G231A, Q232A, and E233A are constructs with single-amino acid substitution at the indicated residue. Hatched bars mark the substitution mutants with the properties obviously different from the authentic control (black bar). (B) Analysis of the substitutability of C228 with structurally similar amino acids. Complementation transformation of the ΔygfZ mutant was done as in (A) except that plumbagin was applied at three different levels. Note: the construct with the Cys to Ser mutation (C228S) behaved indistinguishable from the authentic YgfZ at all different plumbagin amounts applied while C228 M and C228A mutants apparently deviated from the authentic when plumbagin was applied at 100 μg per disc. Insets: exogenous His_x6-tagged YgfZ constructs were expressed in the transformed ΔygfZ strain comparably as revealed by Western blotting; DnaK served as a protein-loading control. Note: pQE60 served as negative control. To compare the significance of the data, results from the authentic YgfZ were used as a reference. NS: no significance; * p < 0.05