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.

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds

“…Recombinant TfuDyP was overproduced in E. coli BL21 as a His6 fusion protein with an Nterminal 40-amino acid putative Tat signal sequence [16] removed. The enzyme was purified by nickel affinity chromatography, and the fusion tag removed using TEV protease, yielding purified protein largely free of contaminants (see Supporting Information Figure S1).…”

“…A DyP-type peroxidase has also been reported from Thermobifida fusca, a thermophilic cellulose degrader, which was shown to have activity towards a range of phenolic and anthraquinone dye substrates, and to catalyse enantioselective sulfoxidation reactions [16]. Since T. fusca will encounter the lignin polymer during cellulose degradation, we wanted to examine whether the T. fusca DyP has peroxidase activity towards lignin.…”

Surface enhanced resonance Raman detection of a catalytic intermediate of DyP-type peroxidase

“…[1][2][3][4][5][6][7][8][9][10] They lack the distal histidine that is highly conserved in classical peroxidases and which acts as an acid/base catalyst in the reduction of hydrogen peroxide to water. DyPs are classified into four phylogenetically distinct classes (A-D); their physiological role is not fully established yet and it appears to be subfamilydependent.…”

“…9 Thus, SERR in particular represents a promising approach for a more detailed mechanistic analysis of DyPs from different subfamilies, stabilizing either Cpd I or Cpd II. [1][2][3][4][5][6][7][8] We have recently observed Cpd I with t 1/2 4 60 h in distal PpDyP variants D132N and N136L, in which aspartate 132 was replaced by asparagine and asparagine 136 by leucine, respectively. 24 Future studies of these systems will contribute to a better understanding of both, molecular determinants of catalytic activity in DyP-type peroxidases, and of their mechanistic properties in the immobilised state, which is a prerequisite for application of these catalytically versatile and efficient enzymes in the construction of bioelectronic devices.…”

First Crystal Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase

“…Lately, other reactions catalyzed by DyPs have been discovered, e.g. the oxidative cleavage of carotenoids (10) and sulfoxidation of aromatic sulfides (11). Although the mentioned and yet to be fully unraveled chemical properties of DyPs demonstrate their obvious biotechnological potential, their actual role in nature remains obscure.…”