细胞色素P450 Kemp消除酶的改造及新型催化机制解析

doi:10.1016/S1872-2067(23)64389-X

催化学报 ›› 2023, Vol. 47: 191-199.DOI: 10.1016/S1872-2067(23)64389-X

细胞色素P450 Kemp消除酶的改造及新型催化机制解析

李爱涛^a^,¹, 汪倩^a^,¹, 宋希彤^b^,¹, 张小栋^a, 黄建文^a, 陈纯琪^a, 郭瑞庭^a,*(), 王斌举^b,*(), Manfred T. Reetz^c^,^d,*()

^a湖北大学生命科学学院, 工业生物技术湖北省重点实验室, 省部共建生物催化与酶工程国家重点实验室, 湖北武汉430062, 中国
^b厦门大学化学化工学院, 理论与计算化学福建省重点实验室, 固体表面物理化学国家重点实验室, 福建厦门360015, 中国
^c马克斯.普朗克煤炭研究所, 慕尼黑, 德国
^d中国科学院天津工业生物技术研究所, 天津300308, 中国

收稿日期:2022-10-23 接受日期:2022-12-09 出版日期:2023-04-18 发布日期:2023-03-20
通讯作者: *电子信箱: guoreyting@hubu.edu.cn (郭瑞庭),wangbinju2018@xmu.edu.cn(王斌举),reetz@mpi-muelheim.mpg.de (M. Reetz).
作者简介:¹共同第一作者.
基金资助:
国家重点研发计划(2019YFA0906400);湖北省生物催化与酶工程技术引智创新示范基地项目(2019BJH021);湖北省自然科学基金创新团体项目(2020CFA011);省部共建生物催化与酶工程国家重点实验室自主研发项目

Engineering of a P450-based Kemp eliminase with a new mechanism

Aitao Li^a^,¹, Qian Wang^a^,¹, Xitong Song^b^,¹, Xiaodong Zhang^a, Jian-Wen Huang^a, Chun-Chi Chen^a, Rey-Ting Guo^a,*(), Binju Wang^b,*(), Manfred T. Reetz^c^,^d,*()

^aState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Key Laboratory of Industrial Biotechnology, School of life science, Hubei University, Wuhan 430062, Hubei, China
^bState Key Laboratory of Physical Chemistry of Solid Sur-faces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 360015, Fujian, China
^cMax-Planck-Institut für Kohlenforschung, 45470 Muelheim, Germany
^dTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

Received:2022-10-23 Accepted:2022-12-09 Online:2023-04-18 Published:2023-03-20
Contact: *E-mail: guoreyting@hubu.edu.cn (R. Guo),wangbinju2018@xmu.edu.cn (B. Wang),reetz@mpi-muelheim.mpg.de (M. Reetz).
About author:¹ Contributed equally to this work.
Dedicated to Prof. Karl-Erich Jaeger on the occasion of his retirement.
Supported by:
National Key Research and Development Program of China(2019YFA0906400);Innovation base for Introducing Talents of Discipline of Hubei Province(2019BJH021);Natural Science Foundation Innovative Group Project of Hubei Province(2020CFA011);Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering

摘要/Abstract

摘要：

传统的Kemp消除反应可以通过氢氧化钾和三烷基胺等碱性物质, 催化底物苯并异恶唑开环生成产物2-氰基苯酚. 三十年来, Kemp消除反应一直被用作模式反应来设计或定向进化新型生物酶催化剂, 从而揭示未知的酶催化机制的复杂性, 增强对酶催化机制的理解. 目前科研人员使用不同的蛋白作为骨架设计能够高效催化Kemp消除反应的人工酶. 例如Hilvert及Mayo等基于人工酶HG3.17, 设计获得了Kemp消除酶, 可以催化5-硝基苯并异恶唑生成产物2-氰基-4-硝基苯酚. 通过17轮定向进化获得的最终突变体展现出与天然酶相近的催化活性(k_cat/K_m = 230000 L mol^‒1 s^‒1; k_cat = 700 s^‒1). 该研究不仅表明蛋白质工程可以进化出高效的生物酶, 量子力学/分子力学(QM/MM)分析还揭示了突变体催化活性提高的分子机制. 与酸碱催化的Kemp消除反应不同, 最近Korendovych等报道以肌红蛋白作为骨架基于氧化还原机制的Kemp消除反应, 通过开发一种独特的基于核磁共振(NMR)的蛋白质定向进化技术, 快速鉴定出热点氨基酸位点并获得了高催化活性的人工酶突变体, 其同样达到了天然酶的催化活性.

此前我们研究(Nat. Commun., 2017, 8, 14876)发现, 与传统的酸碱催化机制完全不同, 细胞色素P450-BM3能够通过氧化还原的机制催化Kemp消除反应. 本文继续以P450-BM3为蛋白骨架, 对其进一步改进以提高催化活性. 以P450-BM3突变体F87G (k_cat = 3.0 s^‒1)为模板, 借助双密码子(酪氨酸和赖氨酸, Y-K)饱和突变策略, 对其活性中心的六个关键氨基酸位点进行组合突变, 经过筛选获得了活性大幅度提高的三突变体F87G/L75Y/T438K (k_cat = 27.4 s^‒1). 为进一步解析其催化活性提高的分子机制, 首先对该突变体与底物复合物的晶体结构进行了解析, 结果发现底物在突变体活性口袋的构象与野生型P450-BM3完全不同, 底物的硝基指向了Heme辅基. 对其进行了QM/MM计算研究, 发现Heme-Fe(II)首先将电子转移到底物并与硝基配位, 随后, 有效促进了底物N‒O键还原裂解, 并使生成的中间产物Heme-Fe(III)-NO₂和苯氧基阴离子更稳定, 最后, 通过键的旋转和质子转移生成产物2-氰基-4-硝基苯酚. 由此可见, 同一P450酶的不同突变体能够以两种不同的底物结合模式以及氧化还原机制来催化Kemp消除反应. 综上, 本文获得了P450酶催化Kemp消除反应构效关系和催化机制新的认识, 为进一步改造该酶提高其催化活性提供借鉴.

关键词: Kemp消除反应, 氧化还原机理, P450单加氧酶, 量子力学/分子力学, X射线结构, 饱和突变

Abstract:

For three decades the biocatalytic version of the Kemp elimination of 5-nitro-benzisoxazole (1) has served as a forum for testing the creation of different artificial enzymes, the primary aim being to reveal mechanistic intricacies and to extend our understanding of enzymatic catalysis as such. In general, acid/base catalysis pertains, but recently a novel redox based mechanism was postulated when using P450-BM3 mutants as scaffolds. In the present study, we report an surprising discovery made upon employing new P450-BM3 variants generated by rational enzyme design, which points to the existence of a new and different redox based mechanism. X-ray structural data and theoretical analyses based on MD simulations and QM/MM calculations support this conclusion. The results of this study are of relevance in the human metabolism of therapeutic drugs and in redox mediated biosynthesis catalyzed by P450s.

Key words: Kemp elimination, Redox mechanism, P450 monooxygenase, Quantum mechanical/molecular, mechanical, X-ray structure, Saturation mutagenesis

李爱涛, 汪倩, 宋希彤, 张小栋, 黄建文, 陈纯琪, 郭瑞庭, 王斌举, Manfred T. Reetz. 细胞色素P450 Kemp消除酶的改造及新型催化机制解析[J]. 催化学报, 2023, 47: 191-199.

Aitao Li, Qian Wang, Xitong Song, Xiaodong Zhang, Jian-Wen Huang, Chun-Chi Chen, Rey-Ting Guo, Binju Wang, Manfred T. Reetz. Engineering of a P450-based Kemp eliminase with a new mechanism[J]. Chinese Journal of Catalysis, 2023, 47: 191-199.

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图/表 8

Fig. 1. Designed enzyme catalyzed Kemp elimination via a Br?nsted acid/base mechanism (B = base; BH = acid) [16].

Fig. 2. Redox based P450-BM3 catalyzed Kemp elimination via two different mechanisms. (a) Previously reported P450-BM3 catalyzed Kemp elimination with a redox-based mechanism [16]. (b) New mechanism of a redox based Kemp eliminase using rationally selected mutants of P450-BM3.

Fig. 3. Engineering of P450-BM3 for Kemp elimination of substrate 1. (a) Zoom of the active site of WT P450-BM3. (b) The present strategy of directed evolution for P450-BM3 with F87G as template. Double code saturation mutagenesis (DCSM) at two randomization sites A (S72, L75, T438) and B (A82, L181, A328), using bulky lysine and tyrosine as the double code K-Y. (c) Michaelis-Menten plots for triple mutant F87G/L75Y/T438K (TMK) (red) and WT (blue). The data represents the average of three independent measurements, with error bars denoting s.d.

Table 1 Summary of kinetic parameters for P450-BM3 and variants catalyzing the Kemp elimination of 1 with different mechanisms. In all cases purified enzymes were used a.

Catalyst	K_m (mmol L^‒1)	k_cat (s^-1)	k_cat/K_m (L s^-1 mol^-1)		Reference
WT P450BM-3	>6	>1.5	240 ± 60	N/A	[17]
F87G	2.5 ± 0.6	3.0 ± 0.5	1,200 ± 200	2.6 × 10⁶
A82F	0.27±0.03	8.4 ± 0.4	31000 ± 1,500	0.7 × 10⁷
F87G/A82F	1.3 ± 0.2	11.5 ± 0.7	8800 ± 700	1.0 × 10⁷
F87G/L75Y	1.7 ± 0.2	4.4 ± 0.2	2600 ± 100	3.8 × 10⁶	This study
F87G/L75Y/T438K	4.7 ± 0.5	27.4 ± 2.2	5800 ± 400	2.4 × 10⁷
F87G/L75Y/T438R	6.5 ± 0.9	28.1 ± 3.3	4300 ± 500	2.4 × 10⁷
F87G/L75Y/T438L	1.6 ± 0.2	9.0 ± 0.2	5600 ± 100	0.8 × 10⁷

Fig. 4. Exploration of deconvoluted mutations in TMK (F87G/L75Y/T438K) as catalysts in the Kemp elimination of substrate 1. (a) Kemp elimination of substrate 1 catalyzed by the P450-BM3 variant TMK (F87G/L75Y/T438K) and by deconvoluted variants thereof with the cell free extract. (b) Kemp elimination of substrate 1 catalyzed by P450-BM3 variants with different amino acid substitutions at position 438 based on the parental variant F87G/L75Y; cell free extracts were used as catalysts.

Fig. 5. Crystal structure of apo- and ligand-bound form of P450-BM3 variant TMK. (a) The overall structure of each crystal is presented in cartoon models, with heme and bound ligands shown in sticks. (b) The protein-ligand interaction networks in TMK-substrate complex. Hemes are shown by purple blue sticks and soaked ligands in tint/magenta sticks. Amino acids are shown in lines, with mutated ones colored in the same scheme as the bound ligands. The yellow dashed lines indicate distance < 3.5 ?.

Fig. 6. Illustration of MD analyses. (a) Non-bonding interactions between the substrate and the protein environment obtained from 50 ns MD simulations. The red line corresponds to the binding conformation in which the nitro group is in close proximity to heme-Fe (Fig. 6(b)), with an average non-bonding interaction value of ?30.8 kcal mol?1. The black line corresponds to the binding conformation in which the N1 atom of substrate is in close proximity to heme-Fe (Fig. 6(c)), with an average non-bonding interaction value of ?26.5 kcal mol?1. (b) The most populated snapshot from clustering of MD trajectory for the substrate binding conformation in which the nitro group is in close proximity to heme-Fe. (c) The most populated snapshot from clustering of MD trajectory for the substrate binding conformation in which the N1 atom of substrate is in close proximity to heme-Fe.

Fig. 7. (a) The calculated mechanism for the TMK-catalyzed Kemp elimination derived from QM/MM simulations. (b) The representative structures of key species obtained from QM/MM simulations (key distances are given in angstroms) (Fig. S9). The spin densities of iron and substrate are given in blue font.

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细胞色素P450 Kemp消除酶的改造及新型催化机制解析

Engineering of a P450-based Kemp eliminase with a new mechanism

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[1]	吴殷琦, 陈倩倩, 陈琦, 耿强, 张巧玉, 郑宇璁, 赵晨, 张龑, 周佳海, 王斌举, 许建和, 郁惠蕾. 精准调控Baeyer-Villiger单加氧酶的底物选择性以避免拉唑亚砜的过氧化[J]. 催化学报, 2023, 51(8): 157-167.
[2]	赵鹤云;赵忠泽;赵义芬;柳清菊;. SnO2 纳米棒的氧化还原特性[J]. 催化学报, 2010, 31(1): 44-48.