Limits of Versatility of Versatile Peroxidase

Abstract

Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn(2+)-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 Mn(2+)-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, Mn(2+), Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn(2+) 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 Mn(2+) only under acidic conditions. We have determined that Mn(2+) inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn(2+) and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn(2+) and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn(2+)-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 Mn(2+) at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn(2+) and pH levels both in the growth medium and in the reaction mixture.

Importance: VP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn(2+) binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi.

FIG 1

UV-vis spectra and SDS-PAGE of the purified P. ostreatus VP1. VP1 shows a typical UV-visible spectrum (270 to 700 nm) with a Soret peak at 406 nm and a high Rz value (A₄₀₆/A₂₈₀) of 5.4. (Inset) VP1 shows a single band on 4 to 12% SDS-PAGE in MES buffer. This band was identified by LC-MS/MS as VP1 with a high purity level of 95% and no evidence of other peroxidases.

FIG 2

pH stability of P. ostreatus VP1. Residual activities after 6 (A) and 24 (B) h of incubation at 8 different pH levels in the range of 2 to 9 are shown. Activities of oxidation of Mn²⁺ (broken), OII (black), and RB5 (gray) are displayed in activity units (U/ml). These activities were determined after preincubation of the enzyme in 100 mM B&R buffer at a suitable pH level. Data represent averages from three biological replicates. Bars denote the standard deviations.

FIG 3

Thermal stability of P. ostreatus VP1. Residual activities after 6 h of incubation at 10 different temperatures in the range of 25 to 70°C are shown. Activities of oxidation of Mn²⁺ (broken), OII (black), and RB5 (gray) are displayed in activity units (U/ml). Activities were determined after preincubation of the enzyme at a suitable temperature. The enzyme was dissolved in 10 mM sodium tartrate buffer at pH 5 or pH 3 to determine Mn²⁺ oxidation or direct oxidations of the dyes, respectively. Data represent averages from three biological replicates. Bars denote the standard deviations.

FIG 4

Optimum pH of P. ostreatus VP1 activities. The oxidations of Mn²⁺, OII, and RB5 were determined using 100 mM sodium tartrate buffer in a range of 2.5 to 7.0. Activities of oxidation of Mn²⁺ (broken), OII (black), and RB5 (gray) are displayed in activity units (U/ml). Data represent averages from three replicates. Bars denote the standard deviations.

FIG 5

In vivo decolorizations occurred only under acidic pH in the absence of Mn²⁺. In vivo decolorization of both OII (right) and RB5 (left) (100 mg/liter) by PC9 (upper) and Δvp1 (lower) strains after 15 days of incubation is depicted. (A) The decolorizations were studied in solid GP medium (2% agar) under two different initial pH conditions, 6 and 3.5. (B) pH monitoring of the agar plates during the incubation period of PC9 (upper) and Δvp1 (lower) strains. GP media at initial pHs of 6 (circle) and 3.5 (square) without supplement of dye (black), with OII (orange), and with RB5 (blue) are shown. These represent at least three biological replicates.

FIG 6

Orange II inhibits Mn²⁺ oxidation by P. ostreatus VP1. Oxidation of 100 μM Mn²⁺ (expressed in values corresponding to the optical density at 238 nm [OD₂₃₈]) in the absence of OII (open black squares). In the presence of 50 μM OII, oxidation of Mn²⁺ (filled black squares) and OII (black triangles) was monitored with the same reaction mixture. The reaction was repeated three independent times.

FIG 7

Effect of Mn²⁺ on Michaelis-Menten saturation curves of direct oxidation (OII or RB5) by VP1. Data represent oxidation of OII in the absence (A) and presence (B) of 0.05 μM Mn²⁺ and oxidation of RB5 in the absence (C) and presence (D) of 0.05 μM Mn²⁺. Each reaction was repeated three independent times.

FIG 8

Proposed pathway for OII oxidation by purified P. ostreatus VP1 in the absence of Mn²⁺. The reaction mixtures contained sodium tartrate, pH 3, 50 μM OII, 0.05 μM enzyme, and 0.1 mM H₂O₂. Samples were analyzed after 1, 5, 10, and 20 min of incubation. All of the transformation products (TPs) were identified in all samples but in different relative amounts (data not shown). Under these conditions, evidence of asymmetric and symmetric cleavages were found. The brackets indicate TPs with proposed structures (the TPs are numbered as described in Table 4).

FIG 9

OII transformation product (TP) accumulation during in vitro reaction with purified P. ostreatus VP1 in the absence of Mn²⁺. The TPs are numbered as described in Table 4. (A) TP3, 6-hydroxyl-1,2 naphthoquinone; (B) TP4, BS (B); (C) TP5, 4HBS; (D) TP7, 3-aminonaphthalene-2,6-dione.