Improved manganese-oxidizing activity of DypB, a peroxidase from a lignolytic bacterium

Abstract

DypB, a dye-decolorizing peroxidase from the lignolytic soil bacterium Rhodococcus jostii RHA1, catalyzes the peroxide-dependent oxidation of divalent manganese (Mn(2+)), albeit less efficiently than fungal manganese peroxidases. Substitution of Asn246, a distal heme residue, with alanine increased the enzyme's apparent k(cat) and k(cat)/K(m) values for Mn(2+) by 80- and 15-fold, respectively. A 2.2 Å resolution X-ray crystal structure of the N246A variant revealed the Mn(2+) to be bound within a pocket of acidic residues at the heme edge, reminiscent of the binding site in fungal manganese peroxidase and very different from that of another bacterial Mn(2+)-oxidizing peroxidase. The first coordination sphere was entirely composed of solvent, consistent with the variant's high K(m) for Mn(2+) (17 ± 2 mM). N246A catalyzed the manganese-dependent transformation of hard wood kraft lignin and its solvent-extracted fractions. Two of the major degradation products were identified as 2,6-dimethoxybenzoquinone and 4-hydroxy-3,5-dimethoxybenzaldehyde, respectively. These results highlight the potential of bacterial enzymes as biocatalysts to transform lignin.

Figure 1

Steady-state kinetic analysis of Mn²⁺ oxidation by N246A. Reactions contained 20 nM N246A and 1 mM H₂O₂ in 50 mM malonate, pH 5.5 at 25 °C. The solid line represents a best fit of the Michaelis-Menten equation to the data using LEONORA.

Figure 2

The active site and Mn²⁺-binding pocket of N246A. Residues (green) and heme (orange) are shown as sticks. The iron (dark orange), solvent species (grey) and Mn²⁺ (magenta) are shown as spheres. The Mn²⁺-binding site is shown with an omit F_o-F_c map (grey mesh) contoured at 4.2σ, and a Mn-anomalous map (red) contoured at 8σ. Figures were made using PyMol.

Figure 3

Incubation of HKL and its fractions with N246A. Reaction mixtures contained 0.25 mg ml⁻¹ of a preparation of HKL, 100 nM N246A, 20 mM MnSO₄, and 0.5 mM H₂O₂ (20 mM sodium malonate, pH 5.5, 15% DMSO) and were incubated at 30 °C. The Panels (A) and (C) show reactions after 10 and 60 min, respectively. Panels (B) and (D) are the corresponding reactions performed in the absence of enzyme. The lignin preparation used in each reaction is identified at the top of each column.

Figure 4

Chromatographic analyses of the Mn²⁺-dependent transformation of HKL with N246A. Reactions were performed for 60 min essentially as described for Figure 3. The soluble products were extracted and analyzed using reverse phase HPLC. The elution profiles of reactions incubated with (red traces) and without (black traces) N246A are shown.

Figure 5

Structural characterization of transformation products obtained from HKL-F1. The mass spectra of the purified products eluting at t_r = 12 min (top) and t_r = 31 min (bottom) are shown. Structures corresponding to the fragmentation patterns are shown in inset.