Limits of versatility of versatile peroxidase

Although Mn²⁺ is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn²⁺-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn²⁺-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn²⁺, Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn²⁺ oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn²⁺ only under acidic conditions. We have determined that Mn²⁺ inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn²⁺ and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn²⁺ and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn²⁺-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn²⁺ at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn²⁺ and pH levels both in the growth medium and in the reaction mixture.