Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis

Abstract

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

Expression and Characterization of a Dye-Decolorizing Peroxidase from Pseudomonas Fluorescens Pf0-1

“…The purified enzyme was most active at pH 4.0 ( Figure 2) with moderate activity between pH 3.0 and 6.0, which is in perfect agreement with other studies on DyPs [9,23,26]. The relative activity of the purified PfDyP B2 enzyme was tested using 0.5 mM ABTS and 0.1 mM hydrogen peroxide in the presence of various cations (Ca 2+ , Mg 2+ , Zn 2+ , Mn 2+ , Co 2+ , Fe 2+ , and Hg 2+ ) and reducing agents (aminotriazole, EDTA, imidazole, DTT, Cys, and Na-azide).…”

“…The purified enzyme was most active at pH 4.0 ( Figure 2) with moderate activity between pH 3.0 and 6.0, which is in perfect agreement with other studies on DyPs [9,23,26].…”

“…Using hydrogen peroxide as an electron acceptor, they are capable of catalyzing efficient oxidations of a wide array of industrially relevant substrates including dyes with anthraquinone structure, β-carotene, and aromatic sulfides [4][5][6][7][8]. Instead of a distal histidine acting as an acid-base catalyst, DyPs contain an aspartate active site residue within a highly conserved GXXDG distal motif [8], although recent findings indicate that instead of aspartate some DyPs have a glutamate [9]. On the proximal side, the heme iron of DyPs is coordinated by a histidine.…”

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases

“…DyP from the alkaliphilic cellulomonad, Cellulomonas bogoriensis (CboDyP) is capable of oxidation of anthraquinone, azo and indigoid dyes, its pH opt is 5 (for Reactive Blue 19, RB19, as oxidizing substrate). 11 The elucidated X-ray structure of CboDyP shows Glu, Arg and Phe in the distal haem cavity; the presence of a Glu instead of conserved Asp, indicates a unique GXXEG distal motif. 11 The high frequency region of RR spectra of CboDyP shows narrow, well resolved core size bands at 1371 (n 4 ), 1481 (n 3 ) and 1561 cm À1 (n 2 ) at neutral and pH opt , Fig.…”

“…Crystal structures have been solved for a number of DyPs, oen at a relatively high pH. [1][2][3][11][12][13][14] However, since the haem spin conguration that governs the H 2 O 2 binding and reactivity is in peroxidases particularly sensitive to pH, temperature, and physical state (solution vs. crystal), 17 it is of crucial importance to understand the conguration of DyPs active site in solution. To that end Resonance Raman (RR) spectroscopy has provided a wealth of information about the haem conguration, structure and catalytic mechanism of classical peroxidases.…”