A Dye-Decolorizing Peroxidase from Vibrio cholerae

Abstract

The dye-decolorizing peroxidase (DyP) protein from Vibrio cholerae (VcDyP) was expressed in Escherichia coli, and its DyP activity was assayed by monitoring degradation of a typical anthraquinone dye, reactive blue 19 (RB19). Its kinetic activity was obtained by fitting the data to the Michaelis-Menten equation, giving kcat and Km values of 1.3 ± 0.3 s(-1) and 50 ± 20 μM, respectively, which are comparable to those of other DyP enzymes. The enzymatic activity of VcDyP was highest at pH 4. A mutational study showed that two distal residues, Asp144 and Arg230, which are conserved in a DyP family, are essential for the DyP reaction. The crystal structure and resonance Raman spectra of VcDyP indicate the transfer of a radical from heme to the protein surface, which was supported by the formation of the intermolecular covalent bond in the reaction with H2O2. To identify the radical site, each of nine tyrosine or two tryptophan residues was substituted. It was clarified that Tyr129 and Tyr235 are in the active site of the dye degradation reaction at lower pH, while Tyr109 and Tyr133 are the sites of an intermolecular covalent bond at higher pH. VcDyP degrades RB19 at lower pH, while it loses activity under neutral or alkaline conditions because of a change in the radical transfer pathway. This finding suggests the presence of a pH-dependent switch of the radical transfer pathway, probably including His178. Although the physiological function of the DyP reaction is unclear, our findings suggest that VcDyP enhances the DyP activity to survive only when it is placed under a severe condition such as being in gastric acid.

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects

“…This channel is proposed to be the entrance of H 2 O 2 and also exists in DyPB and Vc DyP. 35–36 The second heme access channel has a diameter of ~8.0 Å and leads to the heme 6-propionate group (right panel in Figure 2B), which is larger than the corresponding channels in DyPB (~6.0 Å) and Vc DyP (~7.5 Å). 35–36 It has been reported that the propionate group can provide a direct electron transfer path from the porphyrin radical to a bound substrate.…”

Identification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata

“…Additionally, this tyrosine has been proposed as a potential surface-exposed protein radical site in B- and D-class DyPs. 18,19,49 As depicted in Figure 2, structural alignment of Tc DyP (A-class) and Aau DyP (D-class) based on the heme cofactor reveals that the conserved tyrosines in two proteins are overlapped. The same result was obtained when structures of Tc DyP and Vc DyP from Vibrio cholerae (B-class) were aligned (data not shown).…”

Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae

“…3 Briefly, the hutZ gene was subcloned into pET-28b (Merck Millipore, Darmstadt, Germany) via Nde I and EcoR I sites, and the thrombin recognition site (Leu-Val-Pro-Arg-Gly-Ser) in the pET-28b construct was mutated to the HRV 3C protease recognition site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro). 15 E. coli strains transduced with HutZ expression plasmids were grown at 37 °C in LB broth supplemented with 50 μg/mL kanamycin. Expression of His-tagged fusion protein in E. coli BL21(DE3) was induced by adding isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.4 mM, after which cells were further incubated at 28 °C overnight.…”