ChemCatChem
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2022
·Vol. 14
·期 24
·e202201151
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IF 5.497
Fanhui Kong,Jie Chen,Xiangquan Qin,Chuanfei Liu,Yiping Jiang,Li Ma,Huifang Xu,Shengying Li,Zhiqi Cong
Graphical Enzymatic Approach for the Biosynthesis of Indigoid Dyes : A stepwise laboratory evolution of P450 enzyme is developed and used to establish an environmentally benign enzymatic approach for the biosynthesis of indigoid dyes from indole derivatives with high isolation yields, improving upon existing methods. Herein, we describe an environmentally benign enzymatic approach for the preparation of indigoid from indole derivatives. A series of beneficial P450BM3 mutants were obtained using a stepwise approach involving site-directed, random, and combinatory hot-site mutations. Using H 2 O 2 as the terminal oxidant and N -( ω -imidazolyl)-hexanoyl- L -phenylalanine as a co-catalyst, the quadruple-mutant F87G/T268V/F77I/E140D efficiently catalyzed the 3-hydroxylation of indole and subsequent autooxidation to indigo with higher efficiency than the flavin-containing monooxygenase, PTDH-mFMO, which was the best oxidizing enzyme for indigo synthesis reported to date. Following this procedure, 12 indigoid compounds were prepared using indole derivatives with various substituents as starting materials with moderate to high isolated yields (31.3–79.5 %). The catalytic efficiencies ( k cat / K m ) of the beneficial mutants for typical indole substrates ranged from 182–2849 mM −1 ⋅ min −1 , almost 2- to 712-fold higher than those of the reported P450 enzymes. This study provides a new enzymatic approach for the biosynthesis of indigoid dyes from indole derivatives.
研究领域:
生物催化
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酶工程
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有机化学
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绿色化学
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DOI
高影响因子
Nature Communications
·
2025
·Vol. 16
·期 1
·1-12
·
IF 15.7
Yang Liu,Pu Zhongji,Wu Jianping,Liu Xiaofeng,Wang Zhe,Yu Haoran,Wang Liuwei,Meng Yan,Xu Gang,Yang Lirong,Zheng Wenlong
N -oxidation of N -heterocycles is essential in the synthesis of natural products but challenging due to low efficacy and poor regioselectivity. In this study, the N -oxidation selective potential of P450BM3 from Bacillus megaterium for N -heterocyclic compounds is investigated. Here, twelve amino acids located in the active center, including A74, L75, V78, A82, F87, I263, A264, A328, P329, A330, I401, and L437, are investigated by site-saturation mutation. As a result, F87, A264, L75, V78, A328, I401, and L437 are identified as hotspot residues. Subsequently, the combinatorial active-site saturation test/iterative saturation mutagenesis strategy is performed. Using quinoline as a model substrate, the mutant F87G/A264G/A328L exhibits N -oxidation selectivity of up to 99.0%, with a conversion rate of 99.3%. Molecular dynamics simulations uncover a “push-pull” molecular mechanism elucidating the pivotal role of steric factors in determining substrate recognition and N -oxidation selectivity. This study provides an efficient N -oxide synthesis method and insights into P450BM3’s molecular mechanisms.
研究领域:
生物化学
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生物催化
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酶工程
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计算生物学
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有机合成
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产品关联
DOI
PubMed
高影响因子
Molecular Catalysis
·
2025
·
IF 4.9
Xiaojuan Yu,Zili Zhang,Wenlong Lu,Qian Li,Chenghua Gao,Cefan Zhou,Jingfeng Tang,Zhiyong Guo,Aitao Li
Steroids are important pharmaceutically active compounds, and cytochrome P450 monooxygenases (CYPs) serve as promising biocatalysts for steroidal drug synthesis. However, their application is often limited by low catalytic efficiency and incomplete understanding of regioselectivity mechanisms, despite their remarkable oxygen activation capabilities. In this study, we engineered P450BM3 to enable high-efficiency C15α-hydroxylation of d -ethylgonendione, producing a key intermediate in the synthesis of gestodene, a widely used progestogen hormonal contraceptive. Initial screening of a laboratory P450 library identified variant HG12 (P450BM3/F87G/A328G/A330W), which exhibited 54.1 % regioselectivity and 45.8 % substrate conversion. Subsequent semi-rational design targeting residues lining in the active pocket and substrate channel, variant HG12M8 (R47W/F87G/M185L/A328G/A330W/T438G) was obtained, achieving 89.5 % regioselectivity and 90.1 % conversion. Molecular dynamics simulations revealed that F87G and M185L mutations alleviate steric hindrance and stabilize substrate binding, while R47W widens the substrate access channel. This work presents a dual-purpose protein engineering strategy to overcome efficiency and selectivity challenges in P450s while providing a robust biocatalyst for sustainable steroidal pharmaceutical production.
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DOI
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2023
·Vol. 24
·期 3
·2395
·
IF 5.6
Yanqing Li,Yingwu Lin,Fang Wang,Jinghan Wang,Osami Shoji,Jiakun Xu
With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity withkcatof 1402 min−1and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity (kcat/Km= 1388 mM−1min−1) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo.
研究领域:
生物技术
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酶工程
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绿色化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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