Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2

Abstract

Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.

FIG. 1.

Comparison of the microenvironment surrounding the exposed Trp in PcLiPH8 (A and C) and P. ostreatus MnP2 (B and D). The surrounding environments of Trp171 in the PcLiPH8 crystal structure from the PDB code 1B82 (A and C) and those of Trp170 in the P. ostreatus MnP2 3D model (B and D) obtained by homology modeling with PeVPL (PDB code 2BOQ) as a template are exhibited. The RMSD of pairwise Cα atoms of the P. ostreatus MnP2 (VP) and PeVPL was 0.43 Å. The superficial structures are represented in panels A and B; the red and blue colors indicate negative and positive electrostatic potentials, respectively. The molecular structures are shown in panels C and D; the red and blue modules indicate oxygen and nitrogen, respectively, and the dashed green line indicates an H bond.

FIG. 2.

SDS-PAGE analysis before and after deglycosylation. Wild-type MnP2 (lanes 1 and 3) and R263N (lanes 2 and 4), with (lanes 3 and 4) or without (lanes 1 and 2) N-glycosidase treatment, were subjected to SDS-PAGE analysis. The sizes of the molecular mass standards (lane M) are indicated on the right side. The cleavage of N-glycans from the purified enzymes was carried out by glycopeptidase F under denaturing conditions (pH 8.6) at 37°C for 20 h.

FIG. 3.

Oxidation activity of high-molecular-weight substrates by the MnP2 variants. (A) Oxidation of RNase A was evaluated by measuring the relative emission at 410 nm with an excitation wavelength of 315 nm. Reaction mixtures consisted of each variant enzyme (3.0 U/ml) and RNase A (100 μM) in 50 mM sodium tartrate buffer (pH 3.0). Reactions were initiated by the addition of H₂O₂ (60 μM). (B) Decolorization of Poly R-478 was assayed at A_520/350. Reaction mixtures consisted of each enzyme (3.0 U/ml) and 0.02% Poly R-478, in 50 mM sodium tartrate buffer (pH 3.0). Reactions were initiated by the addition of 0.2 mM H₂O₂. Symbols: ○, wild-type Mn2; ▪, W170A; •, R263N; ▵, Q266F; ▴, V166/168L. The averages and standard deviations were determined from three independent experiments.

FIG. 4.

Effect of preincubation at pH 3.0 on the reactivity with different substrates in wild-type MnP2 and R263N. Wild-type MnP2 and R263N were preincubated in 50 mM sodium tartrate buffer (pH 3.0) for 30, 60, 90, or 120 min, followed by measurement of the reactivity with Poly R-478 (A), Mn²⁺(B), and VA (C). The relative activity of the enzyme for each substrate was evaluated by taking the maximum reactivity as 100%. Symbols: ○, wild-type Mn2; •, R263N. The averages and standard deviations were determined from three independent experiments.

FIG. 5.

pH optima of wild-type MnP2 and R263N for different substrates. The pH optima of reactivity for Poly R-478 (A), Mn²⁺(B), and VA (C) were surveyed by using wild-type MnP2 and R263N in 50 mM sodium tartrate buffer (pH 2.0 to 5.0). The relative activity of the enzyme for each substrate was evaluated by taking the maximum reactivity as 100%. Symbols: ○, wild-type Mn2; •, R263N. The averages and standard deviations were determined from three independent experiments.

FIG. 6.

Spontaneous reduction of the compound I form of MnP2 variants and MnP3. UV spectra for compound I formation and its spontaneous decay in the absence of a substrate were measured. Compound I was obtained by adding one equivalent of H₂O₂ to the reaction mixture, and its self-reduction was monitored. Increases in absorbance traces at 418 and 407 nm show reduction to compound II and a resting enzyme, respectively. Traces 1 to 11 correspond to 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 min, respectively, and trace 0 indicates the spectrum before H₂O₂ addition (resting state). The typical spectra for the resting state (RS), compound I (CI), and compound II (CII) are indicated in the figure.

FIG. 7.

Surface conformation around the redox active Trp residue in PcLiPH8 and P. ostreatus MnP2 variants. Views from the upper side of the redox active Trp residue in PcLiPH8 and P. ostreatus MnP2 variants are displayed. The surface of Trp residue is colored green and indicated by an arrow. The yellow-colored residues indicate the introduced amino acid substitution (Q266F and V166L). V168L was buried inside and was invisible from the surface. The 3D structure of PcLiPH8 was from PDB code 1B82, and those of P. ostreatus MnP2 variants were obtained by homology modeling with PeVPL (PDB code 2BOQ) as a template. The RMSDs of pairwise Cα atoms of Q266F and PeVPL and of V166/168L and PeVPL were 0.45 and 0.44 Å, respectively. The red and blue colors indicate negative and positive electrostatic potentials, respectively.