Engineering substrate recognition sites of cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber for enhanced regiospecific naphthalene hydroxylation

Sha Tao; Yang Gao; Kang Li; Qiuhao Lu; Chenggang Qiu; Xin Wang; Kequan Chen; Pingkai Ouyang

doi:10.1016/j.mcat.2020.111089

Engineering substrate recognition sites of cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber for enhanced regiospecific naphthalene hydroxylation

Sha Tao, Yang Gao, Kang Li, Qiuhao Lu, Chenggang Qiu, Xin Wang, Kequan Chen, Pingkai Ouyang

COLLEGE OF BIOTECHNOLOGY AND PHARMACEUTICAL ENGINEERING

Nanjing Tech University

Research output: Contribution to journal › Article › peer-review

14 Scopus citations

Abstract

In this study, a cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber was engineered to convert naphthalene selectively into 1-naphthol. The substrate recognition sites (SRSs) of CYP116B3 were identified using structure-based computational analysis. Subsequently, site-directed and saturation mutagenesis were conducted on SRS1, SRS2, and SRS3. The improved resulting triple mutant (E88C-N199Q-Q209A) was characterized. A final 1-naphthol titer of 8.26 mg/L/h was achieved, which is 14-fold higher than the control. The docking results suggest that the conformational changes produced by directed evolution improved the substrate binding environment. The results presented here hold promise for 1-naphthol production by P450 enzymes.

Original language	English
Article number	111089
Journal	Molecular Catalysis
Volume	493
DOIs	https://doi.org/10.1016/j.mcat.2020.111089
State	Published - Sep 2020

Keywords

Molecular docking simulation
P450 monooxygenase
Polycyclic aromatic hydrocarbon
Protein engineering

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10.1016/j.mcat.2020.111089

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title = "Engineering substrate recognition sites of cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber for enhanced regiospecific naphthalene hydroxylation",

abstract = "In this study, a cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber was engineered to convert naphthalene selectively into 1-naphthol. The substrate recognition sites (SRSs) of CYP116B3 were identified using structure-based computational analysis. Subsequently, site-directed and saturation mutagenesis were conducted on SRS1, SRS2, and SRS3. The improved resulting triple mutant (E88C-N199Q-Q209A) was characterized. A final 1-naphthol titer of 8.26 mg/L/h was achieved, which is 14-fold higher than the control. The docking results suggest that the conformational changes produced by directed evolution improved the substrate binding environment. The results presented here hold promise for 1-naphthol production by P450 enzymes.",

keywords = "Molecular docking simulation, P450 monooxygenase, Polycyclic aromatic hydrocarbon, Protein engineering",

author = "Sha Tao and Yang Gao and Kang Li and Qiuhao Lu and Chenggang Qiu and Xin Wang and Kequan Chen and Pingkai Ouyang",

note = "Publisher Copyright: {\textcopyright} 2020 Elsevier B.V.",

year = "2020",

month = sep,

doi = "10.1016/j.mcat.2020.111089",

language = "英语",

volume = "493",

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T1 - Engineering substrate recognition sites of cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber for enhanced regiospecific naphthalene hydroxylation

AU - Tao, Sha

AU - Gao, Yang

AU - Li, Kang

AU - Lu, Qiuhao

AU - Qiu, Chenggang

AU - Wang, Xin

AU - Chen, Kequan

AU - Ouyang, Pingkai

PY - 2020/9

Y1 - 2020/9

N2 - In this study, a cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber was engineered to convert naphthalene selectively into 1-naphthol. The substrate recognition sites (SRSs) of CYP116B3 were identified using structure-based computational analysis. Subsequently, site-directed and saturation mutagenesis were conducted on SRS1, SRS2, and SRS3. The improved resulting triple mutant (E88C-N199Q-Q209A) was characterized. A final 1-naphthol titer of 8.26 mg/L/h was achieved, which is 14-fold higher than the control. The docking results suggest that the conformational changes produced by directed evolution improved the substrate binding environment. The results presented here hold promise for 1-naphthol production by P450 enzymes.

AB - In this study, a cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber was engineered to convert naphthalene selectively into 1-naphthol. The substrate recognition sites (SRSs) of CYP116B3 were identified using structure-based computational analysis. Subsequently, site-directed and saturation mutagenesis were conducted on SRS1, SRS2, and SRS3. The improved resulting triple mutant (E88C-N199Q-Q209A) was characterized. A final 1-naphthol titer of 8.26 mg/L/h was achieved, which is 14-fold higher than the control. The docking results suggest that the conformational changes produced by directed evolution improved the substrate binding environment. The results presented here hold promise for 1-naphthol production by P450 enzymes.

KW - Molecular docking simulation

KW - P450 monooxygenase

KW - Polycyclic aromatic hydrocarbon

KW - Protein engineering

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U2 - 10.1016/j.mcat.2020.111089

DO - 10.1016/j.mcat.2020.111089

M3 - 文章

AN - SCOPUS:85087379796

SN - 2468-8231

VL - 493

JO - Molecular Catalysis

JF - Molecular Catalysis

M1 - 111089

ER -

Engineering substrate recognition sites of cytochrome P450 monooxygenase CYP116B3 from Rhodococcus ruber for enhanced regiospecific naphthalene hydroxylation

Abstract

Keywords

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