Abstract
Il-MnP1, a short-type manganese peroxidase from Irpex lacteus F17, can oxidize some substrates in the absence of Mn2+, but the catalysis was much lower than in the presence of Mn2+. Here, we report a mutant R70V/E166A of Il-MnP1 with some unique properties, which possessed clearly higher catalysis for the decolorization of anthraquinone and azo dyes in the absence of Mn2+ than that of Il-MnP1. Importantly, the optimum pH of R70V/E166A for decolorization of anthraquinone dyes (Reactive Blue 19, RB19) was 6.5, and the mutant achieved high decolorization activities in the range of pH 4.0–7.0, whereas Il-MnP1 only showed decolorization for RB19 at pH 3.5–4.0. In addition, the optimum H2O2 concentration of R70V/E166A for RB19 decolorization was eight times that of Il-MnP1 and the H2O2 stability has improved 1.4 times compared with Il-MnP1. Furthermore, Mn2+ competitively inhibited the oxidation of RB19 by R70V/E166A, explaining the higher catalytic activity of the mutant R70V/E166A in the absence of Mn2+. Molecular docking results suggested that RB19 binds to the distal side of the heme plane in mutant R70V/E166A, which extended from the heme δ-side to the heme γ-side, and close to the mutated residues of R70V and E166A, whereas RB19 could not access the heme pocket of Il-MnP1 due to the steric hindrance of the side-chain group of Arg 70. Thus, this study constructed a useful mutant R70V/E166A and analyzed its higher Mn2+-independent activity, which is very important for better understanding the Mn2+-independent catalytic mechanism for short manganese peroxidases.
Key points
• The mutant R70V/E166A of atypical MnP1 of I. lacteus F17 shows unique catalytic properties.
• At pH 6.5, the R70V/E166A had a strong ability to decolorize anthraquinone dyes in the absence of Mn 2+ .
• The binding sites of Reactive Blue 19 in mutant R70V/E166A were elucidated.
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Acknowledgments
A portion of this work was performed on the Steady High Magnetic Field Facilities, High Magnetic Field Laboratory, CAS. Molecular Docking simulation was supported by WECOMPUT Technology Co. (Bei**g, China).
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This research is supported by the National Natural Science Foundation of China (31970100).
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RJ designed the research; JY constructed the mutant R70V/E166A; JLW conducted the biochemistry experiments and analyzed the results; WHH and WTH conducted part of this work. RJ and JLW co-wrote the manuscript. All authors approved the final version of the manuscript.
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Wang, J., Yang, J., Huang, W. et al. A mutant R70V/E166A of short manganese peroxidase showing Mn2+-independent dye decolorization. Appl Microbiol Biotechnol 107, 2303–2319 (2023). https://doi.org/10.1007/s00253-023-12438-y
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DOI: https://doi.org/10.1007/s00253-023-12438-y