The catalytic mechanism of A-type dye-decolourising peroxidase BsDyP: neither aspartate nor arginine is individually essential for peroxidase activity

Abstract

ARTICLE This journal isBsDyP from Bacillus subtilis belongs to the new dye-decolourising peroxidase (DyP) family. Here we use transient kinetics to provide details on the catalytic cycle of BsDyP. The reaction of BsDyP with H 2 O 2 exhibits saturation behaviour consistent with a two-step mechanism involving the formation of an E-H 2 O 2 intermediate (K 1 = (12 ± 1) × 10 -6 M) followed by formation of Compound I (k 1 = 22 ± 1 s -1 ). We demonstrate that the k 1obs is pH-dependent and controlled by an ionisable group with a pK a of 4.3 suggesting the involvement of distal Asp. The reaction of Compound I with guaiacol obeys second order kinetics (k 3 ' = (0.21 ± 0.01) × 10 6 M -1 s -1 ) while the reaction of Compound II with guaiacol shows saturation kinetics (K 4 = 22 ± 5) × 10 -6 M and k 4 = 0.13 ± 0.01 s -1 ) and is the rate-limiting step in the BsDyP catalytic cycle. We furthermore use transient and steady-state kinetics, spectroscopic and electrochemical approaches to investigate the role of distal Asp240, Arg339 and Asn244 and proximal Asp383 residues in BsDyP. All mutations of distal residues affect particularly the K 1 (and K m ) for H 2 O 2 leading to catalytic efficiencies (k cat /K m ) of only one to two orders of magnitude lower than in the wild type. Notably, a significant improvement in the catalytic efficiency for reducing substrates is observed in variants. We conclude that the Asp and Arg residues are important for the proper binding of H 2 O 2 to the haem but none is individually indispensable for promoting H 2 O 2 (de)protonation and O-O bond cleavage. The obtained kinetic data suggest an important role of the distal Asn in modulating the acidbase catalysis of BsDyP. Our findings contribute to the establishment of structural determinants of DyPs that underlie their mechanistic properties; this has implications for their potential in biotechnological applications and sheds more light on subfamily-dependent features of these enzymes.

Serial Femtosecond Zero Dose Crystallography Captures a Water‐Free Distal Heme Site in a Dye‐Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV=O Formation

“…Given that protonation is ap recursor for the oxo species to act as ap otent oxidant, the combination of Arg and Asn as Hbonding donors must tune the pK a of the oxo in DtpB to result in astable,unreactive compound I. Compound I t 1/2 times for the bacterial DyPs from Rhodococuus jostii RHA1 [17a] and Bacillus subtilis, [23] which both possess ad istal Asn, are reported to be 540 sa nd 3.2 h, respectively.T herefore,t he SFX structure of the Fe IV = OD tpB illustrates that the interaction of the Asn with the oxo group must, amongst other factors,s erve to stabilize compound I. In contrast, Asp152 is not H-bonded to the oxo group,with the O d atoms being > 4.4 apart (Table S3).…”

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices

“…Heme-containing peroxidases, including the best studied HRP, as well as cytochrome c peroxidase (C c P), chloroperoxidase and lactoperoxidase, show a significant potential as biocatalysts for the development of 3 rd -generation biotechnological devices [ 29 , 30 ]. Bacterial DyPs are also regarded as promising candidates, since they possess broad substrate specificity, they can easily be heterologously overexpressed in non-glycosylated form, and their properties improved by protein engineering [ 1 , 2 , 31 , 32 , 33 , 34 ]. However, the progress in the development of peroxidase-based applications is lagging behind the expectations, due to the difficulties in transferring the enzyme catalytic efficiency in solution to the immobilised state and the lack of experimental approaches for fast screening of enzyme/electrode constructs in situ [ 30 ].…”

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

“…8 This however may not be the case in all DyPs, as it was demonstrated that neither Asp nor Arg is individually essential for peroxidase activity of Bacillus subtilis DyP (BsDyP). 9 The electrons for H 2 O 2 reduction are provided by the (bulky) substrates that bind close to the haem pocket or at the enzyme surface (for electron delivery via long-range electron transfer). 4,10 Based on phylogenetic analysis and catalytic and structural characteristics, DyPs have been classied into four subfamilies.…”