Characterization of Dye-Decolorizing Peroxidases from Rhodococcus jostii RHA1

The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower...

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Bibliographic Details
Published in:Biochemistry (Easton) 2011-06, Vol.50 (23), p.5108-5119
Main Authors: Roberts, Joseph N, Singh, Rahul, Grigg, Jason C, Murphy, Michael E. P, Bugg, Timothy D. H, Eltis, Lindsay D
Format: Article
Language:English
Subjects:
Anthraquinones - chemistry
Anthraquinones - metabolism
Coloring Agents - chemistry
Coloring Agents - metabolism
Electron Spin Resonance Spectroscopy
Kinetics
Oxidation-Reduction
Peroxidases - chemistry
Peroxidases - metabolism
Rhodococcus - enzymology
Rhodococcus - metabolism
Spectrophotometry, Ultraviolet
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Summary:The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower peroxidase activities than C- and D-type DyPs. Nevertheless, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (k cat/K m = 12800 ± 600 M–1 s–1) than either ABTS or pyrogallol, consistent with previously characterized DyPs. By contrast, DypB showed the greatest apparent specificity for ABTS (k cat/K m = 2000 ± 100 M–1 s–1) and also oxidized MnII (k cat/K m = 25.1 ± 0.1 M–1 s–1). Further differences were detected using electron paramagnetic resonance (EPR) spectroscopy: while both DyPs contained high-spin (S = 5/2) FeIII in the resting state, DypA had a rhombic high-spin signal (g y = 6.32, g x = 5.45, and g z = 1.97) while DypB had a predominantly axial signal (g y = 6.09, g x = 5.45, and g z = 1.99). Moreover, DypA reacted with H2O2 to generate an intermediate with features of compound II (FeIVO). By contrast, DypB reacted with H2O2 with a second-order rate constant of (1.79 ± 0.06) × 105 M–1 s–1 to generate a relatively stable green-colored intermediate (t 1/2 ∼ 9 min). While the electron absorption spectrum of this intermediate was similar to that of compound I of plant-type peroxidases, its EPR spectrum was more consistent with a poorly coupled protein-based radical than with an [FeIVO Por•]+ species. The X-ray crystal structure of DypB, determined to 1.4 Å resolution, revealed a hexacoordinated heme iron with histidine and a solvent species occupying axial positions. A solvent channel potentially provides access to the distal face of the heme for H2O2. A shallow pocket exposes heme propionates to the solvent and contains a cluster of acidic residues that potentially bind MnII. Insight into the structure and function of DypB facilitates its engineering for the improved degradation of lignocellulose.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi200427h