FIG 7.

Phylogenetic analysis of genes from the lower VPP pathway and characterization of VppE. (A) Phylogenetic tree of nucleotide sequences of the gene cluster from vppH to vppG constructed using MEGA6. (B) Enzymatic activity of VppE. The enzyme activity was measured at 320 nm with the addition of 0.34 mg ml⁻¹ cell extract, 250 μM 2,5-DHP, and 250 μM Fe²⁺ (FeCl₂). a, negative control, 2,5-DHP; b, negative control, 2,5-DHP plus Fe²⁺; c, negative control, cell extract containing VppE plus 2,5-DHP; d, negative control, control cell extract plus 2,5-DHP plus Fe²⁺; e, reactions of VppE (cell extract containing VppE plus 2,5-DHP plus Fe²⁺). The concentrations of 2,5-DHP and Fe²⁺ were 250 μM. The control cell extract was from E. coli BL21(DE3) cells with pET28a. The cell extract containing VppE was prepared from E. coli BL21(DE3) cells with pET28a-vppE. (C) HPLC analysis of 2,5-DHP conversion reactions at 0 min and 20 min with cell extract containing VppE (top) and control cell extract (bottom). All the reactions were performed in 50 mM Tris-HCl, pH 8.0, at 25°C.