Open AccessIdentification of Surface-Exposed Protein Radicals and A Substrate Oxidation Site in A-Class Dye-Decolorizing Peroxidase from Thermomonospora curvata
Author(s) -
Ruben Shrestha,
Xuejie Chen,
Kasra X. Ramyar,
Zahra Hayati,
Eric A. Carlson,
Stefan H. Bossmann,
Likai Song,
Brian V. Geisbrecht,
Ping Li
Publication year - 2016
Publication title -
acs catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.898
H-Index - 198
ISSN - 2155-5435
DOI - 10.1021/acscatal.6b01952
Subject(s) - chemistry , radical , active site , peroxidase , lignin , substrate (aquarium) , catalysis , electron paramagnetic resonance , reactive intermediate , photochemistry , organic chemistry , enzyme , oceanography , physics , nuclear magnetic resonance , geology
Dye-decolorizing peroxidases (DyPs) are a family of heme peroxidases, in which a catalytic distal aspartate is involved in H 2 O 2 activation to catalyze oxidations in acidic conditions. They have received much attention due to their potential applications in lignin compound degradation and biofuel production from biomass. However, the mode of oxidation in bacterial DyPs remains unknown. We have recently reported that the bacterial Tc DyP from Thermomonospora curvata is among the most active DyPs and shows activity toward phenolic lignin model compounds ( J. Biol. Chem. 2015 , 290 , 23447). Based on the X-ray crystal structure solved at 1.75 Å, sigmoidal steady-state kinetics with Reactive Blue 19 (RB19), and formation of compound II-like product in the absence of reducing substrates observed with stopped-flow spectroscopy and electron paramagnetic resonance (EPR), we hypothesized that the Tc DyP catalyzes oxidation of large-size substrates via multiple surface-exposed protein radicals. Among 7 tryptophans and 3 tyrosines in Tc DyP consisting of 376 residues for the matured protein, W263, W376, and Y332 were identified as surface-exposed protein radicals. Only the W263 was also characterized as one of surface-exposed oxidation sites. SDS-PAGE and size-exclusion chromatography demonstrated that W376 represents an off-pathway destination for electron transfer, resulting in the crosslinking of proteins in the absence of substrates. Mutation of W376 improved compound I stability and overall catalytic efficiency toward RB19. While Y332 is highly conserved across all four classes of DyPs, its catalytic function in A-class Tc DyP is minimal possibly due to its extremely small solvent accessible areas. Identification of surface-exposed protein radicals and substrate oxidation sites is important for understanding DyP mechanism and modulating its catalytic functions for improved activity on phenolic lignin.