A Dye-Decolorizing Peroxidase from <i>Vibrio
cholerae</i>
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Abstract
The dye-decolorizing peroxidase (DyP)
protein from <i>Vibrio
cholerae</i> (<i>Vc</i>DyP) was expressed in <i>Escherichia coli</i>, 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 <i>k</i><sub>cat</sub> and <i>K</i><sub>m</sub> values of 1.3 ± 0.3 s<sup>–1</sup> and 50
± 20 μM, respectively, which are comparable to those of
other DyP enzymes. The enzymatic activity of <i>Vc</i>DyP
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 <i>Vc</i>DyP 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 H<sub>2</sub>O<sub>2</sub>. 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. <i>Vc</i>DyP 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 <i>Vc</i>DyP enhances the DyP activity to survive only when it
is placed under a severe condition such as being in gastric acid