蛋白重折叠策略整合蛋白骨架与氯化血红素用于构建人工金属酶

doi:10.1016/S1872-2067(24)60150-6

摘要/Abstract

摘要：

人工金属酶(ArMs)由起催化作用的金属催化剂和提供酶促反应微环境的蛋白骨架构成. 人工金属酶的设计着重通过对现存蛋白骨架的理性设计将金属催化剂嵌入其中, 并通过蛋白工程进一步提高催化性能. 该方法受限于蛋白骨架的三维结构, 无法将同一策略广泛地应用于不同的蛋白结构. 同时, 针对每个蛋白骨架的结构分析和精密设计限制了人工金属酶的功能范围拓展和催化效率提高.

本文报道了一种泛用性的设计理念, 通过蛋白重折叠过程将氯化血红素(hemin)深入整合至蛋白骨架中, 打破了传统方法中主要针对蛋白与催化剂接触面的设计修饰, 制得具有高活性的多功能ArMs复合物. 首先, 将苯甲醛裂合酶(BAL)溶于盐酸胍溶液中使蛋白质变性并暴露出所有可反应的活性位点. 随后, 通过巯基-烯点击反应将hemin与蛋白序列中的巯基连接, 并通过重折叠过程将hemin包埋进蛋白的二级结构中, 经过镍柱纯化得到ArMs复合物(hemin@BAL). 热重分析结果表明, 新的复合物结构中具有明显的hemin成分, 并进一步通过电感耦合等离子体原子发射光谱确定了平均每个酶分子含有3.92个hemin分子, 超出了常规化学修饰对于BAL表面巯基(2个)的修饰极限, 表现出相较于传统方法的优越性. 紫外-可见光谱结果表明, ArMs复合物中hemin的吸收峰迁移至410 nm, 说明hemin的金属中心与纯化过程中加入的咪唑分子形成了稳定的螯合. 同时, 扫描透射电子显微镜和能量色散X射线谱结果表明, ArMs复合物呈现为聚集体形态并且表现出一致的碳元素和铁元素分布, 再次证明了hemin与蛋白骨架的稳定结合. 以氧化反应作为模式反应, ArMs在同等hemin含量下表现出3倍于游离hemin的转化率, 并且在动力学分析中表现出远高于天然的辣根过氧化物酶的催化效率. 此外, 该ArMs还表现出等同于化学催化剂hemin的热稳定性, 说明本文策略构建的ArMs具有较好的理化性质. 经过条件优化, ArMs可与葡萄糖氧化酶组成级联反应, 并在60 min内使底物几乎完全氧化. 在此基础上, 该ArMs设计策略对不同的蛋白骨架, 包括南极假丝酵母脂肪酶B和绿色荧光蛋白, 表现出了同等的适用性, 得到了具有高度氧化活性的ArMs复合物. 为进一步探索ArMs的应用潜力, 采用更具挑战性的环丙烷化反应作为模式反应, 并对ArMs的制备过程进行优化, 得到hemin包埋程度更高、螯合更稳定的ArMs复合物(hemin@BAL-opt). 优化后的ArMs催化环丙烷化转化, 经过18 h达到转化率83.7± 3.3%, 远高于游离hemin (1.5 ± 0.4%)和蛋白-hemin混合物(8.0 ± 0.6%).

综上, 本文发展了一种打破传统ArMs设计方法的技术理念, 通过蛋白质变性-光催化点击-重折叠过程将hemin融入蛋白骨架, 得到了一系列具有全新结构的ArMs, 并对不同的蛋白骨架展现出泛用性和一致的催化氧化及环丙烷化的活性提升. 该策略为设计新型人工酶并实现多功能生物催化应用提供了新思路.

关键词: 人工酶, 蛋白质变性, 点击反应, 蛋白重折叠, 氯化血红素

Abstract:

In this study, we unveil a conceptual technology for fabricating artificial metalloenzymes (ArMs) by deeply integrating hemin into protein scaffolds via a protein refolding process, a method that transcends the conventional scope of surface-level modifications. Our approach involves denaturing proteins, such as benzaldehyde lyase, green fluorescent protein, and Candida antarctica lipase B, to expose extensive reactive amino acid residues, which are then intricately linked with hemin using orthogonal click reactions, followed by protein refolding. This process not only retains the proteins’ structural integrity but expands proteins’ functionality. The most notable outcome of this methodology is the hemin@BAL variant, which demonstrated a remarkable 83.7% conversion rate in cyclopropanation reactions, far surpassing the capabilities of traditional hemin-based catalysis in water. This success highlights the significant role of protein structure in the ArMs’ activity and marks a substantial leap forward in chemical modification of proteins. Our findings suggest vast potentials of protein refolding approaches for ArMs across various catalytic applications, paving the way for future advancements in synthetic biology and synthetic chemistry.

Key words: Artificial metalloenzyme, Protein denaturation, Click reaction, Protein refolding, Hemin

欧阳敬平, 张振方, 吴昌柱. 蛋白重折叠策略整合蛋白骨架与氯化血红素用于构建人工金属酶[J]. 催化学报, 2024, 67: 157-165.

Jingping Ouyang, Zhenfang Zhang, René Hübner, Henrik Karring, Changzhu Wu. Artificial metalloenzymes enabled by combining proteins with hemin via protein refolding[J]. Chinese Journal of Catalysis, 2024, 67: 157-165.

×
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(24)60150-6

https://www.cjcatal.com/CN/Y2024/V67/I12/157

图/表 7

Scheme 1. Schematic illustration of ArMs fabrication and their application in oxidation and cyclopropanation reactions.

Fig. 1. The characterization of ArMs. (a) Schematic representation of hemin@BAL ArMs. UV-vis spectral analysis (b), CD spectroscopic profile (c), and TGA data (d) for hemin, BAL, and hemin@BAL. (e) STEM image and corresponding EDXS-based element distribution maps of hemin@BAL, with specific highlighting of C and Fe in red and green color, respectively.

Fig. 2. Catalytic performance of hemin and hemin@BAL in oxidation reactions. All reactions were carried out in triplicate. (a) Reaction scheme of pyrogallol oxidation. (b) Initial reaction profile of pyrogallol (10 mmol L?1) with H2O2 (40 mmol L?1) in a 50 mmol L?1 pH 7.4 PB solution, catalyzed by 33.3 μmol L?1 hemin and 8.5 μmol L?1 hemin@BAL (batch b1). (c) Catalytic activity of hemin@BAL under varied temperature conditions (20, 30, and 40 °C) and different pH levels (6, 7.4, and 8) in PB solutions. (d) The thermostability of hemin and hemin@BAL (batch b1) after incubation at 50 °C. (e) The conversion courses of pyrogallol (10 mmol L?1) with H2O2 (40 mmol L?1) catalyzed by 33.3 μmol L?1 hemin and 8.5 μmol L?1 hemin@BAL (batch b1) in a 50 mmol L?1 pH = 8 PB solution at 40 °C. For more details, please see the Supplementary Information.

Fig. 3. Cascade reactions catalyzed by GOx-ArMs. (a) Schematic overview of the GOx-ArM cascade reaction. (b) Reaction profiles of GOx-b1 under varied experimental conditions: Condition 1 (Con1) ? 0.5 mg mL?1 GOx with 100 mmol L?1 D-glucose. Condition 2 (Con2) ? 0.5 mg mL?1 GOx with 400 mmol L?1 D-glucose. Condition 3 (Con3) ? 1.5 mg mL?1 GOx with 400 mmol L?1 D-glucose.

Fig. 4. Refolding of other proteins for ArMs. (a) UV-vis spectral analysis for hemin and hemin@CalB. (b) CD profiles of hemin and hemin@CalB. (c) Initial reaction profile of hemin and hemin@CalB at 8.5 μmol L?1 level. (d) STEM image and corresponding EDXS-based element distribution maps of hemin@CalB, with specific highlighting of C and Fe in red and green color, respectively. (e) UV-vis spectra for hemin and hemin@GFP. (f) CD profiles of hemin and hemin@GFP. (g) Initial reaction profile of hemin and hemin@GFP at 8.5 μmol L?1 level. (h) STEM image and corresponding EDXS-based element distribution maps of hemin@GFP, utilizing the same color scheme for element representation as applied for hemin@CalB. All reactions were carried out in triplicate.

Fig. 5. Optimized hemin@BAL (hemin@BAL-opt, denoted as opt.). (a) UV-vis spectra of hemin@BAL-opt and hemin. (b) CD spectra of hemin@BAL-opt and BAL. (c) STEM image and corresponding EDXS-based element distribution maps of hemin@BAL-opt, with specific highlighting of C and Fe in red and green color, respectively.

Table 1 Cyclopropanation reaction catalyzed by ArMs.

Entry	Catalyst	Conversion (%)
1	BAL	0
2	hemin	1.5 ± 0.4
3	BAL + hemin	8.0 ± 0.6
4	hemin@BAL (b1)	21.3 ± 3.1
5	hemin@BAL-opt.	83.7 ± 3.3

参考文献

[1]	S. V. Athavale, K. Chen, F. H. Arnold, in: Transition Metal‐Catalyzed Carbene Transformations, J. Wang, C.-M. Che, M. P. Doyle eds., Wiley-VCH GmbH, Weinheim, 2022, 95-138.
[2]	M. Scheffen, D. G. Marchal, T. Beneyton, S. K. Schuller, M. Klose, C. Diehl, J. Lehmann, P. Pfister, M. Carrillo, H. He, S. Aslan, N. S. Cortina, P. Claus, D. Bollschweiler, J.-C. Baret, J. M. Schuller, J. Zarzycki, A. Bar-Even, T. J. Erb, Nat. Catal., 2021, 4, 105-115.
[3]	T. Vornholt, F. Christoffel, M. M. Pellizzoni, S. Panke, T. R. Ward, M. Jeschek, Sci. Adv., 2021, 7, eabe4208.
[4]	S. Hanreich, E. Bonandi, I. Drienovská ChemBioChem, 2023, 24, e202200566.
[5]	D. Hilvert, Annu. Rev. Biochem., 2013, 82, 447-470.
[6]	H. J. Davis, T. R. Ward, ACS Cent. Sci., 2019, 5, 1120-1136.
[7]	F. Schwizer, Y. Okamoto, T. Heinisch, Y. Gu, M. M. Pellizzoni, V. Lebrun, R. Reuter, V. Köhler, J. C. Lewis, T. R. Ward, Chem. Rev., 2018, 118, 142-231.
[8]	C. Mayer, ChemBioChem, 2019, 20, 1357-1364.
[9]	C. Van Stappen, Y. Deng, Y. Liu, H. Heidari, J.-X. Wang, Y. Zhou, A. P. Ledray, Y. Lu, Chem. Rev., 2022, 122, 11974-12045.
[10]	Z. Zhou, G. Roelfes, Nat. Catal., 2020, 3, 289-294.
[11]	D. L. Trudeau, D. S. Tawfik, Curr. Opin. Biotechnol., 2019, 60, 46-52.
[12]	M. Ali, H. M. Ishqi, Q. Husain, Biotechnol. Bioeng., 2020, 117, 1877-1894.
[13]	A. D. McDonald, P. M. Higgins, A. R. Buller, Nat. Commun., 2022, 13, 5242.
[14]	G. W. Anderson, J. E. Zimmerman, F. M. Callahan, J. Am. Chem. Soc., 1964, 86, 1839-1842.
[15]	J.-T. Wang, Y. Hong, X. Ji, M. Zhang, L. Liu, H. Zhao, J. Mater. Chem. B, 2016, 4, 4430-4438.
[16]	L. Zhang, W. Zhao, X. Liu, G. Wang, Y. Wang, D. Li, L. Xie, Y. Gao, H. Deng, W. Gao, Biomaterials, 2015, 64, 2-9.
[17]	J. Nicolas, V. S. Miguel, G. Mantovani, D. M. Haddleton, Chem. Commun., 2006, 4697-4699.
[18]	J. C. Peeler, B. F. Woodman, S. Averick, S. J. Miyake-Stoner, A. L. Stokes, K. R. Hess, K. Matyjaszewski, R. A. Mehl, J. Am. Chem. Soc., 2010, 132, 13575-13577.
[19]	A. Stein, A. D. Liang, R. Sahin, T. R. Ward, J. Organomet. Chem., 2022, 962, 122272.
[20]	J. Serrano-Plana, C. Rumo, J. G. Rebelein, R. L. Peterson, M. Barnet, T. R. Ward, J. Am. Chem. Soc., 2020, 142, 10617-10623.
[21]	W. Gao, W. Liu, T. Christensen, M. R. Zalutsky, A. Chilkoti, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 16432-16437.
[22]	W. Gao, W. Liu, J. A. Mackay, M. R. Zalutsky, E. J. Toone, A. Chilkoti, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 15231-15236.
[23]	T. Himiyama, Y. Okamoto, Molecules, 2020, 25, 2989.
[24]	T. Ueno, T. Koshiyama, S. Abe, N. Yokoi, M. Ohashi, H. Nakajima, Y. Watanabe, J. Organomet. Chem., 2007, 692, 142-147.
[25]	N. Stephanopoulos, M. B. Francis, Nat. Chem. Biol., 2011, 7, 876-884.
[26]	L. D. Cabrita, S. P. Bottomley, Biotechnol. Annu. Rev., 2004, 10, 31-50.
[27]	A. Singh, V. Upadhyay, A. K. Panda, in: Insoluble Proteins: Methods and Protocols, E. García-Fruitós ed.ed., Humana Press, New York, 2015, 283-291.
[28]	R. E. Bird, S. A. Lemmel, X. Yu, Q. A. Zhou, Bioconjugate Chem., 2021, 32, 2457-2479.
[29]	N. B. Cramer, C. N. Bowman,, in: Chemoselective and Bioorthogonal Ligation Reactions, W. R.Algar, P. E.Dawson, I.L. Medintz ed., WILEY-VCH Verlag GmbH & Co. KgaA, Weinheim, 2017, 117-145.
[30]	C. E. Hoyle, C. N. Bowman, Angew. Chem. Int. Ed., 2010, 49, 1540-1573.
[31]	S. L. Scinto, D. A. Bilodeau, R. Hincapie, W. Lee, S. S. Nguyen, M. Xu, C. W. am Ende, M. G. Finn, K. Lang, Q. Lin, J. P. Pezacki, J. A. Prescher, M. S. Robillard, J. M. Fox, Nat. Rev. Methods Primers, 2021, 1, 30.
[32]	B. Morandi, A. Dolva, E. M. Carreira, Org. Lett., 2012, 14, 2162-2163.
[33]	A. Maraite, T. Schmidt, M. B. Ansorge-Schumacher, A. M. Brzozowski, G. Grogan, Acta Crystallogr. Sect. F, 2007, 63, 546-548.
[34]	T. Schmidt, M. Zavrel, A. Spieß, M. B. Ansorge-Schumacher, Bioorg. Chem., 2009, 37, 84-89.
[35]	T. G. Mosbacher, M. Mueller, G. E. Schulz, FEBS J., 2005, 272, 6067-6076.
[36]	P. T. Wingfield, Curr. Protoc. Protein Sci., 2016, 84, A.3A.1-A.3A.8.
[37]	K. L. Maxwell, D. Bona, C. Liu, C. H. Arrowsmith, A. M. Edwards, Protein Sci., 2003, 12, 2073-2080.
[38]	C. Asher, K. A. de Villiers, T. J. Egan, Inorg. Chem., 2009, 48, 7994-8003.
[39]	K. A. de Villiers, C. H. Kaschula, T. J. Egan, H. M. Marques, J. Biol. Inorg. Chem., 2007, 12, 101-117.
[40]	S. Dell’Acqua, E. Massardi, E. Monzani, G. Di Natale, E. Rizzarelli, L. Casella, Int. J. Mol. Sci., 2020, 21, 7553.
[41]	E. Karnaukhova, S. Rutardottir, M. Rajabi, L. Wester Rosenlöf, A. I. Alayash, B. Åkerström, Front. Physiol., 2014, 5, 465.
[42]	Q. Wang, Z. Yang, X. Zhang, X. Xiao, C. K. Chang, B. Xu, Angew. Chem. Int. Ed., 2007, 46, 4285-4289.
[43]	N. J. Greenfield, Nat. Protoc., 2006, 1, 2876-2890.
[44]	A. Micsonai, F. Wien, L. Kernya, Y.-H. Lee, Y. Goto, M. Réfrégiers, J. Kardos, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, E3095-E3103.
[45]	A. Jones, M. A. Zeller, S. Sharma, Prog. Biomater., 2013, 2, 12.
[46]	C. Joshi, P. Kumar, B. Behera, A. Barras, S. Szunerits, R. Boukherroub, S. L. Jain, RSC Adv., 2015, 5, 100011-100017.
[47]	Z.-X. Liang, H.-Y. Song, S.-J. Liao, J. Phys. Chem. C, 2011, 115, 2604-2610.
[48]	M. Khoshouei, R. Danev, J. M. Plitzko, W. Baumeister, J. Mol. Biol., 2017, 429, 2611-2618.
[49]	G. Bharathi, D. Nataraj, S. Premkumar, M. Sowmiya, K. Senthilkumar, T. D. Thangadurai, O. Y. Khyzhun, M. Gupta, D. Phase, N. Patra, S. N. Jha, D. Bhattacharyya, Sci. Rep., 2017, 7, 10850.
[50]	P. C. Ooi, M. A. S. Mohammad Haniff, M. F. Mohd Razip Wee, B. T. Goh, C. F. Dee, M. A. Mohamed, B. Y. Majlis, Sci. Rep., 2019, 9, 6761.
[51]	S. M. Siegel, B. Z. Siegel, Nature, 1958, 181, 1153-1154.
[52]	P. V. Iyer, L. Ananthanarayan, Process Biochem., 2008, 43, 1019-1032.
[53]	C. Silva, M. Martins, S. Jing, J. Fu, A. Cavaco-Paulo, Crit. Rev. Biotechnol., 2018, 38, 335-350.
[54]	Z. M. Galbács, L. J. Csányi, J. Chem. Soc., Dalton Trans., 1983, 2353-2357.
[55]	A. B. Hart, Nature, 1949, 163, 876-877.
[56]	P. Turner, G. Mamo, E. N. Karlsson, Microb. Cell Fact., 2007, 6, DOI: 10.1186/1475-2859-6-9.
[57]	O. P. Ward, M. Moo-Young, Biotechnol. Adv., 1988, 6, 39-69.
[58]	J. Miłek, Braz. J. Chem. Eng., 2018, 35, 995-1004.
[59]	E. Yazıcı, H. Deveci, in: Proceedings of the XIIth. International Mineral Processing Symposium, Mining Engineering Department, Hacettepe University, Ö. Y. Gülsoy, L. Ş. Ergün, N. M. Can, İ. B. Çelik ed., Ankara, 2010, 609-616.
[60]	F. Rudroff, M. D. Mihovilovic, H. Gröger, R. Snajdrova, H. Iding, U. T. Bornscheuer, Nat. Catal., 2018, 1, 12-22.
[61]	J. A. Bauer, M. Zámocká, J. Majtán, V. Bauerová-Hlinková Biomolecules, 2022, 12, 472.
[62]	K. Kleppe, Biochemistry, 1966, 5, 139-143.
[63]	S. Dutta Banik, M. Nordblad, J. M. Woodley, G. H. Peters, ACS Catal., 2016, 6, 6350-6361.
[64]	M. W. Larsen, U. T. Bornscheuer, K. Hult, Protein Expr. Purif., 2008, 62, 90-97.
[65]	S. J. Remington, Protein Sci., 2011, 20, 1509-1519.
[66]	M. Zimmer, Chem. Rev., 2002, 102, 759-782.
[67]	F. Yang, L. G. Moss, G. N. Phillips, Nat. Biotechnol., 1996, 14, 1246-1251.
[68]	Y. Xie, J. An, G. Yang, G. Wu, Y. Zhang, L. Cui, Y. Feng, J. Biol. Chem., 2014, 289, 7994-8006.
[69]	M. Steinhagen, A. Gräbner, J. Meyer, A. E. W. Horst, A. Drews, D. Holtmann, M. B. Ansorge-Schumacher, J. Mol. Catal. B, 2016, 133, S179-S187.
[70]	S. Adhikari, R. Crehuet, J. M. Anglada, J. S. Francisco, Y. Xia, Proc. Natl. Acad. Sci. U. S. A., 2020, 117, 18216-18223.
[71]	N. J. Bongiardina, S. M. Soars, M. Podgorski, C. N. Bowman, Polym. Chem., 2022, 13, 3991-4003.
[72]	J. W. Smalley, A. J. Birss, R. Withnall, J. Silver, Biochem. J., 2002, 362, 239-245.
[73]	J. W. Smalley, J. Silver, P. J. Marsh, A. J. Birss, Biochem. J., 1998, 331, 681-685.
[74]	L. Villarino, K. E. Splan, E. Reddem, L. Alonso-Cotchico, C. Gutiérrez de Souza, A. Lledós, J.-D. Maréchal, A.-M. W. H. Thunnissen, G. Roelfes, Angew. Chem. Int. Ed., 2018, 57, 7785-7789.