Artificial metalloenzymes enabled by combining proteins with hemin via protein refolding

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

Abstract

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

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.

×
share this article

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(24)60150-6

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

Figures/Tables 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

References

[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.