电子转移途径中芳香族氨基酸的引入提高了细胞色素P450s的催化性能

doi:10.1016/S1872-2067(23)64445-6

催化学报 ›› 2023, Vol. 49: 81-90.DOI: 10.1016/S1872-2067(23)64445-6

电子转移途径中芳香族氨基酸的引入提高了细胞色素P450s的催化性能

孟帅奇^a^,¹, 李忠玉^b^,¹, 季宇^a^,^*^,¹(), Anna Joelle Ruff^a, 刘珞^b, Mehdi D. Davari^c^,^*(), Ulrich Schwaneberg^a^,^d^,^*()

^a亚琛工业大学生物技术研究所, 德国
^b北京化工大学北京生物过程重点实验室, 北京 100029, 中国
^c莱布尼茨植物生物化学研究所生物有机化学系, 德国
^d莱布尼茨互动材料研究所, 德国

收稿日期:2023-03-29 接受日期:2023-04-10 出版日期:2023-06-18 发布日期:2023-05-06
通讯作者: ^*电子信箱: yu.ji@biotec.rwth-aachen.de (Y. Ji),Mehdi.Davari@ipb-halle.de (M. D. Davari),u.schwaneberg@biotec.rwth-aachen.de (U. Schwaneberg).
作者简介:第一联系人：
¹共同第一作者.
基金资助:
中国留学基金委员会(201906880011)

Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s

Shuaiqi Meng^a^,¹, Zhongyu Li^b^,¹, Yu Ji^a^,^*^,¹(), Anna Joelle Ruff^a, Luo Liu^b, Mehdi D. Davari^c^,^*(), Ulrich Schwaneberg^a^,^d^,^*()

^aInstitute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany
^bBeijing Bioprocess Key Laboratory, Beijing University of Chemical Technology, Beijing 100029, China
^cDepartment of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, Halle06120, Germany
^dDWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, Aachen52074, Germany

Received:2023-03-29 Accepted:2023-04-10 Online:2023-06-18 Published:2023-05-06
Contact: ^*E-mail: yu.ji@biotec.rwth-aachen.de (Y. Ji), Mehdi.Davari@ipb-halle.de (M. D. Davari), u.schwaneberg@biotec.rwth-aachen.de (U. Schwaneberg).
About author:First author contact:¹Contributed equally to this work.
Supported by:
Ph.D. Scholarship from the China Scholarship Council(201906880011)

摘要/Abstract

摘要：

细胞色素P450s是用于生物合成的多功能催化剂. 在P450催化循环中, 需要两个电子来还原血红素铁, 并通过电子转移途径(eTPs)激活随后的还原, 该步骤是反应的限速步骤. 本文重新设计了巨大芽孢杆菌P450 BM3的eTPs, 大幅提高了其催化性能. 通过在P450 BM3的eTP中引入芳香族氨基酸, “最佳”变体P2H02(A399Y/Q403F)的催化效率比P450 BM3野生型催化效率提高了12.9倍(k_cat/K_M从65.8 L mol^‒1 s^‒1提高到913.5 L mol^‒1 s^‒1). 分子动力学模拟和电子传递分析表明, 在辅因子FMN和血红素之间引入的芳香族氨基酸可以显著提高电子转移速率和酶催化性能. 同时, 在电子传递途径中引入酪氨酸可以保护P450的催化中心, 使其避免被氧化性中间产物所破坏, 从而提高其催化效率. 此外, 引入芳香族氨基酸的策略被证明对其他P450(如CYP116B3)同样有效, 改造后的酶表现出显著提高的催化效率. 综上, 本文策略有望拓展到其他带有长程电子传递链的氧化还原酶上, 成为改造的通用策略.

关键词: 定向进化, P450 BM3, 蛋白质工程, 电子传递, 理性设计

Abstract:

Cytochrome P450s are versatile catalysts for biosynthesis applications. In the P450 catalytic cycle, two electrons are required to reduce the heme iron and activate the subsequent reductions through proposed electron transfer pathways (eTPs), which often represent the rate-limiting step in reactions. Herein, the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs. By introducing aromatic amino acids on eTPs of P450 BM3, the “best” variant P2H02 (A399Y/Q403F) showed 13.9-fold improved catalytic efficiency (k_cat/K_M = 913.5 L mol^‒1 s^‒1) compared with P450 BM3 WT (k_cat/K_M = 65.8 L mol^‒1 s^‒1). Molecular dynamics simulations and electron hopping pathways analysis revealed that aromatic amino acid substitutions bridging the cofactor flavin mononucleotide and heme iron could increase electron transfer rates and improve catalytic performance. Moreover, the introduction of tyrosines showed positive effects on catalytic efficiency by potentially protecting P450 from oxidative damage. In essence, engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s (such as CYP116B3) and could be expanded to other oxidoreductases relying on long-range electron transfer pathways.

Key words: Directed evolution, P450 BM3, Protein engineering, Electron transfer, Rational design

孟帅奇, 李忠玉, 季宇, Anna Joelle Ruff, 刘珞, Mehdi D. Davari, Ulrich Schwaneberg. 电子转移途径中芳香族氨基酸的引入提高了细胞色素P450s的催化性能[J]. 催化学报, 2023, 49: 81-90.

Shuaiqi Meng, Zhongyu Li, Yu Ji, Anna Joelle Ruff, Luo Liu, Mehdi D. Davari, Ulrich Schwaneberg. Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s[J]. Chinese Journal of Catalysis, 2023, 49: 81-90.

×
扫码分享

导出引用管理器 EndNote|Ris|BibTeX

链接本文: https://www.cjcatal.com/CN/10.1016/S1872-2067(23)64445-6

https://www.cjcatal.com/CN/Y2023/V49/I6/81

图/表 5

Fig. 1. The selected residues on putative eTPs between cofactor FMN and heme iron of P450 BM3 (PDB ID: 1BVY (16)) for SSM. The FMN and heme domains are shown in the orange and green cartoons, respectively. The targeted amino acid residues are shown as cyan sticks.

Table 1 The kinetic parameters, coupling efficiency, and total turnover number (TTN) of P450 BM3 WT and seven improved variants from TDB library. The values were obtained from triplicate measurements. P1H11: F390Y/A399Y/Q403F; P1G03: F390Y/A399F/Q403Y; P1H02: A399F/Q403F; P1H09: A399F/Q403Y; P2H02: A399Y/Q403F; P2G03: F390Y/A399F/Q403F; P3A04: F390Y/A399F/Q403W.

Variant	K_M (mmol L^‒1)	k_cat (s^‒1)	k_cat/K_M (L mol^‒1 s^‒1)	Coupling efficiency	TTN*
WT	10.95 ± 0.80	0.72 ± 0.05	65.8	31.18 ± 0.97	1196 ± 70
P1H11 (F390Y/A399Y/Q403F)	2.31 ± 0.18	1.13 ± 0.10	489.2	48.25 ± 1.53	1984 ± 27
P1G03 (F390Y/A399F/Q403Y)	3.26 ± 0.38	1.18 ± 0.01	362.0	47.41 ± 0.39	2332 ± 131
P1H02 (A399F/Q403F)	8.61 ± 0.16	1.14 ± 0.06	132.4	47.92 ± 1.22	2560 ± 28
P1H09 (A399F/Q403Y)	6.51 ± 0.28	1.28 ± 0.03	196.6	49.81 ± 2.99	2207 ± 62
P2H02 (A399Y/Q403F)	1.04 ± 0.21	0.95 ± 0.03	913.5	47.61 ± 2.50	2582 ± 108
P2G03 (F390Y/A399F/Q403F)	1.66 ± 0.51	1.14 ± 0.01	686.7	44.33 ± 1.68	2615 ± 85
P3A04 (F390Y/A399F/Q403W)	3.21 ± 0.88	1.21 ± 0.09	376.9	37.97 ± 1.42	2174 ± 70

Fig. 2. The superexchange efficiencies between the cofactor FMN and heme iron of P450 BM3 WT and improved variants from TDB library. (a) The edge-to-edge distances between the cofactor FMN and heme iron determined from 50 ns MD simulations. The MD simulations were run in triplicate (run_1, run_2, run_3) and shown with orange, brown, and blue lines, respectively. (b) The time-averaged distances between the cofactor FMN and heme iron (blue column) and corresponding ET rate (orange scatter) of P450 BM3 WT and beneficial variants. The distances were averaged over the last 40 ns from three independent MD runs. The ET rates were calculated by equation (2) utilizing the time-averaged distance. (c) Comparison of total turnover number (TTN) and the time-averaged distance between the cofactor FMN and heme iron of P450 WT and beneficial variants. The dashed line shows the linear fit to the scattered points. (d) Comparison of coupling efficiency and the time-averaged distance between the cofactor FMN and heme iron of P450 WT and beneficial variants. The dashed line shows the linear fit to the scattered points.

Fig. 3. Predicted electron hopping pathways between the cofactor FMN and heme iron of P450 BM3: P450 BM3 WT (a) and variant P2H02 (b). The results are visualized in 3D (left) and 2D (right). The electron hopping directions are shown in black arrows. In 2D visualization, the center-of-mass distances (in ?) between neighboring residues are shown in purple.

Fig. 4. Proposed rescue pathway of P450 BM3 and variant P2H02. (a) The schematic diagram of oxidative damage of P450 BM3. The red explosions repent the potential oxidation compound. The probable antioxidant rescue pathways from the cofactor heme to the surface of P450 BM3 WT (b) and variant P2H02 (c). The pathways of P450 BM3 were indicated by colors in grey, and the new pathway in variant P2H02 was indicated by colors in orange. The arrows showed the hole transport directions. τM represented the exact mean residence time, which is defined as the inverse of the effective start-to-finish transfer rate [39]. Low τM value represents the fast hole hopping rate.

参考文献

[1]	S. T. Jung, R. Lauchli, F. H. Arnold, Curr. Opin. Biotechnol., 2011, 22, 809-817.
[2]	Z. Li, Y. Jiang, F. P. Guengerich, L. Ma, S. Li, W. Zhang, J. Biol. Chem., 2020, 295, 833-849.
[3]	R. Bernhardt, J. Biotechnol., 2006, 124, 128-145.
[4]	C. J. Whitehouse, S. G. Bell, L. L. Wong, Chem. Soc. Rev., 2012, 41, 1218-1260.
[5]	B. E. Eser, Y. Zhang, L. Zong, Z. Guo, Chin. J. Chem. Eng., 2021, 30, 121-135.
[6]	S. Meng, J. Guo, K. Nie, T. Tan, H. Xu, L. Liu, ChemBioChem, 2019, 20, 2232-2235.
[7]	T. Kubo, M. W. Peters, P. Meinhold, F. H. Arnold, Chem. Eur. J., 2006, 12, 1216-1220.
[8]	S. Grobe, C. P. S. Badenhorst, T. Bayer, E. Hamnevik, S. Wu, C. Grathwol, A. Link, S. Koban, H. Brundiek, B. Grossjohann, U. T. Bornscheuer, Angew. Chem. Int. Ed., 2020, 60, 753-757.
[9]	Y. Watanabe, S. Laschat, M. Budde, O. Affolter, Y. Shimada, V. B. Urlacher, Tetrahedron, 2007, 63, 9413-9422.
[10]	S. Meng, Y. Ji, L. Zhu, G. V. Dhoke, M. D. Davari, U. Schwaneberg, Biotechnol. Adv., 2022, 61, 108051.
[11]	L. H. Xu, Y. L. Du, Synth. Syst. Biotechnol., 2018, 3, 283-290.
[12]	H. M. Girvan, A. W. Munro, Curr. Opin. Chem. Biol., 2016, 136-145.
[13]	A. W. Munro, D. G.Leys, K. J. McLean, K. R. Marshall, T. W. B. Ost, S. Daff, C. S. Miles, S. K. Chapman, D. A. Lysek, C. C. Moser, C. C. Page, P. L. Dutton, Trends Biochem. Sci., 2002, 27, 250-257.
[14]	C. C. Chen, J. Min, L. Zhang, Y. Yang, X. Yu, R.-T. Guo, Chembiochem, 2021, 22, 1317-1328.
[15]	K. D. Dubey, S. Shaik, J. Am. Chem. Soc., 2018, 140, 683-690.
[16]	I. F. Sevrioukova, H. Li, H. Zhang, J. A. Peterson, T. L. Poulos, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 1863-1868.
[17]	R. Verma, U. Schwaneberg, D. Roccatano, Biopolymers, 2014, 101, 197-209.
[18]	D. Darimont, M. J. Weissenborn, B. A. Nebel, B. Hauer, Bioelectrochemistry, 2018, 119, 119-123.
[19]	M. Blanusa, A. Schenk, H. Sadeghi, J. Marienhagen, U. Schwaneberg, Anal. Biochem., 2010, 406, 141-146.
[20]	J. Nazor, U. Schwaneberg, ChemBioChem, 2006, 7, 638-644.
[21]	S. Meng, Y. Ji, L. Liu, M. D. Davari, U. Schwaneberg, ChemSusChem, 2021, 15, e202102434.
[22]	T. Omura, R. Sato, J. Biol. Chem., 1964, 239, 2370-2378.
[23]	W. L. DeLano, The PyMOL molecular graphics system. http://www.pymol.org, 2002.
[24]	H. Land, M. S. Humble, Methods Mol. Biol., 2018, 1685, 43-67.
[25]	E. Krieger, R. L. Dunbrack, R. W. Hooft, B. Krieger, Comput. Drug Discovery Des., 2012, 819, 405-421
[26]	D. A. Case, V. Babin, J. T. Berryman, P. A. Kollman, AMBER 14;University of California: San Francisco, 2014, 1-826.
[27]	J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case, J. Comput. Chem., 2004, 25, 1157-1174.
[28]	A. Jakalian, D. B. Jack, C. I. Bayly, J. Comput. Chem., 2002, 23, 1623-1641.
[29]	P. Mark, L. Nilsson, J. Phys. Chem. A, 2001, 105, 9954-9960.
[30]	O. Trott, A. J. Olson, J. Comput. Chem., 2010, 31, 455-461.
[31]	R. A. Marcus, N. Sutin, BBA-Rev. Bioenergetic, 1985, 811, 265-322.
[32]	M. Cordes, B. Giese, Chem. Soc. Rev., 2009, 38, 892-901.
[33]	Y. D. Ivanov, A. Taldaev, A. V. Lisitsa, E. A. Ponomarenko, A. I. Archakov, Molecules, 2022, 27, 1386.
[34]	M. Cordes, A. Kottgen, C. Jasper, O. Jacques, H. Boudebous, B. Giese, Angew. Chem. Int. Ed., 2008, 47, 3461-3463.
[35]	B. Giese, M. Wang, J. Gao, M. Stoltz, P. Muller, M. Graber, J. Org. Chem., 2009, 74, 3621-3625.
[36]	J. G. Nathanael, L. F. Gamon, M. Cordes, P. R. Rablen, T. Bally, K. M. Fromm, B. Giese, U. Wille, ChemBioChem, 2018, 19, 922-926.
[37]	R. N. Tazhigulov, J. R. Gayvert, M. Wei, K. B. Bravaya, J. Phys. Chem. B, 2019, 123, 6946-6951.
[38]	D. N. Beratan, J. Betts, J. Onuchic, Science, 1991, 252, 1285-1288.
[39]	R. D. Teo, R. Wang, E. R. Smithwick, A. Migliore, M. J. Therien, D. N. Beratan, Proc. Natl. Acad. Sci. U. S. A., 2019, 116, 15811-15816.
[40]	M. Tavanti, F. Parmeggiani, J. R. G. Castellanos, A. Mattevi, N. J. Turner, ChemCatChem, 2017, 9, 3338-3348.
[41]	L. Chen, R. Tang, Z. Li, S. Liang, Bull. Korean Chem. Soc., 2012, 33, 459-463.
[42]	A. Shah, B. Adhikari, S. Martic, A. Munir, S. Shahzad, K. Ahmad, H.-B. Kraatz, Chem. Soc. Rev., 2015, 44, 1015-1027.
[43]	J. B. Derr, J. Tamayo, J. A. Clark, M. Morales, M. F. Mayther, E. M. Espinoza, K. Rybicka-Jasinska, V. I. Vullev, Phys. Chem. Chem. Phys., 2020, 22, 21583-21629.
[44]	C. C. Page, C. C. Moser, X. Chen, P. L. Dutton, Nature, 1999, 402, 47-52.
[45]	B. Giese, S. Eckhardt, M. Lauz, Encyclopedia of Radicals in Chemistry, Biology and Materials, 2012.
[46]	H. B. Gray, J. R. Winkler, Acc. Chem. Res., 2018, 51, 1850-1857.
[47]	J. R. Winkler, H. B. Gray, Philos. Trans. A Math. Phys. Eng. Sci., 2015, 373, 1-7.
[48]	H. B. Gray, J. R. Winkler, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 10920-10925.
[49]	H. B. Gray, J. R. Winkler, Chem. Sci., 2021, 42, 13998-14003.
[50]	J. R. Winkler, H. B. Gray, Q. Rev. Biophys., 2015, 48, 411-420.
[51]	Y. V. Grinkova, I. G. Denisov, M. A. McLean, S. G. Sligar, Biochem. Biophys. Res. Commun., 2013, 430, 1223-1227.
[52]	L. Liu, R. D. Schmid, V. B. Urlacher, Biotechnol. Lett., 2010, 32, 841-845.

电子转移途径中芳香族氨基酸的引入提高了细胞色素P450s的催化性能

Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s

RichHTML

PDF

Supporting Information

可视化

摘要/Abstract

引用本文

使用本文

扫码分享

图/表 5

参考文献

相关文章 5

编辑推荐

Metrics

[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]	周紫璇, 高鹏. 多相催化CO₂加氢直接合成大宗化学品与液体燃料[J]. 催化学报, 2022, 43(8): 2045-2056.
[3]	曹宇飞, 戈钧. 酶复合催化剂原位合成新方法及生物催化应用[J]. 催化学报, 2021, 42(10): 1625-1633.
[4]	林建新, 张留明, 王自庆, 王榕, 魏可镁. Pr 掺杂对 Ru/CeO₂ 催化剂结构和氨合成性能的影响[J]. 催化学报, 2012, 33(3): 536-542.
[5]	赵博;陶进;马吉胜;廉虹;王岩;常琳;高仁钧;曹淑桂. 定向进化提高枯草芽孢杆菌脂肪酶的活力[J]. 催化学报, 2009, 30(4): 291-296.