Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects

Abstract

Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs’ bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme’s adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs’ acidic pH optimum. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs’ lignolytic activity.

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

“…Similar to classes A and C/D ( 1 , 2 ), all described B-class DyPs ( Rodococcus jostii ( 6 , 7 ), Pseudomonas putida ( 8 ), Escherichia coli ( 9 ), Vibrio cholerae ( 10 ), and Enterobacter lignolyticus ( 11 )) exhibit peroxidase activities with conventional substrates (2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, pyrogallol, veratryl alcohol, hydroquinones, etc. ), antraquinone-derived dyes ( e.g.…”

“…), antraquinone-derived dyes ( e.g. Reactive Blue 19), Mn(II) ions, or even lignin model compounds ( 6 , 10 – 13 ). However, compared with other heme peroxidase families, the reported k cat / K m values are relatively small, thus raising the question of in vivo electron donors.…”

“…However, the distinct functional role of the distal catalytic amino acids in the heterolytic cleavage of H 2 O 2 is still under dispute. In B-class DyPs, both the Arg (DyPs from R. jostii RHA1 ( 7 ) and P. putida ( 8 )) and the Asp (Dyp from E. lignolyticus , El DyP ( 11 )) have been proposed to act as proton acceptor and donor in Compound I formation. In A and C/D classes, the distal Asp was suggested to act as acid–base catalyst ( 3 , 17 ).…”

“…Likewise, the mechanism of Compound I reduction and the electronic features of Compound II of B-class DyPs remain elusive. Based on studies of solvent isotope and viscosity effects on Compound I reduction of El DyP, it was proposed that the reducing substrate binds to Compound I instead of free enzyme and mediates its two-electron reduction without formation of an oxoiron(IV)-type Compound II ( 11 ).…”

Biochemical features of dye‐decolorizing peroxidases: Current impact on lignin degradation

“…The two domains, alpha and beta, form the active site cavity that hosts heme. To date, 39 crystal structures of DyPs are known (Table 1) [64,70,71,72,75,[77][78][79][80][81][82][83][84][85][86][87][88][89][90]. Detailed studies of the catalytic cycle show that hydrogen peroxide deprotonation is the first step (Fig.…”