A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily

Abstract

DyP-type peroxidases comprise a novel superfamily of heme-containing peroxidases which is unrelated to the superfamilies of known peroxidases and of which only a few members have been characterized in some detail. Here, we report the identification and characterization of a DyP-type peroxidase (TfuDyP) from the thermophilic actinomycete Thermobifida fusca. Biochemical characterization of the recombinant enzyme showed that it is a monomeric, heme-containing, thermostable, and Tat-dependently exported peroxidase. TfuDyP is not only active as dye-decolorizing peroxidase as it also accepts phenolic compounds and aromatic sulfides. In fact, it is able to catalyze enantioselective sulfoxidations, a type of reaction that has not been reported before for DyP-type peroxidases. Site-directed mutagenesis was used to determine the role of two conserved residues. D242 is crucial for catalysis while H338 represents the proximal heme ligand and is essential for heme incorporation. A genome database analysis revealed that DyP-type peroxidases are frequently found in bacterial genomes while they are extremely rare in other organisms. Most of the bacterial homologs are potential cytosolic enzymes, suggesting metabolic roles different from dye degradation. In conclusion, the detailed biochemical characterization reported here contributes significantly to our understanding of these enzymes and further emphasizes their biotechnological potential.

Fig. 1

Export of TfuDyP to the periplasm by the Tat system in E. coli. a Alignment of TfuDyP and the sequences of YwbN and YcdB from B. subtilis and E. coli, respectively. Solid bars under the sequences indicate the Tat-signal sequence in which the two arginines of the twin-arginine motif are highlighted by two asterisks. For clarity, only residues conserved in all proteins are shaded. The conserved GXXDG motif is boxed, and residues typically conserved amongst DyP-type peroxidases are displayed by asterisks. b Tat-dependent periplasmic transport of TfuDyP was investigated in wild-type (WT) MC4100 cells and strain B1LK0 (ΔTatC). E. coli cells expressing TfuDyP were fractionated into total cells (T), cytoplasm (C), and periplasm (P). Samples were normalized on the basis of OD₆₆₀ and analyzed by immunoblotting with the indicated antisera. Black lines indicate that intervening lanes have been spliced out. The precursor (black dot) and mature form (arrowhead) are indicated

Fig. 2

TfuDyP contains non-covalently bound heme. a MC1061 cells expressing TfuDyP were fractionated into a cell extract (CE) and soluble fraction (Sol), containing cytoplasmic and periplasmic proteins. TfuDyP was isolated from the soluble fraction by Ni²⁺–NTA agarose purification as described in “Materials and methods”. To monitor the purification procedure, samples were taken and analyzed by SDS-PAGE followed by protein staining of the gel. FT flow through, W1 and W2 first and second wash fractions, E eluate. The precursor (black dot) and mature form (arrowhead) are indicated. b UV-visible spectra of oxidized (ox) and reduced (red) TfuDyP. Spectra were recorded with the purified enzyme in PBS at ambient temperature. TfuDyP was reduced by addition of sodium dithionite. The inset shows the pyridine hemochrome spectrum of TfuDyP after reduction with dithionite in pyridine as described in “Materials and methods”

Fig. 3

TfuDyP is a bona fide DyP-type peroxidase. a Influence of pH on Reactive Blue 19-decolorizing activity of TfuDyP. The activity of TfuDyP with Reactive Blue 19 was determined in 25 mM citrate buffer adjusted to different pH values and containing 100 μM H₂O₂. b Identification of enzymatically active forms of TfuDyP. Purified TfuDyP was analyzed on a 7.5% native PAGE gel, and one part of the gel was stained with Coomassie Brilliant Blue (CBB), and the other part was stained for peroxidase activity, using 3,3-diaminobenzidine (DAB). The apo-precursor (asterisk), precursor (black dot), and mature form (greater than symbol) are indicated. c Role of D242 and H338 in enzymatic activity and heme binding. UV-visible spectra of purified TfuDyPD242A (D242A), TfuDyPH338A (H338A), and the wild-type enzyme (WT). Spectra were recorded with the enzyme in PBS at ambient temperature