Research Progress of Artificial Metalloenzymes

doi:10.13523/j.cb.2304002

China Biotechnology

2023, Vol. 43

Issue (10): 72-84 DOI: 10.13523/j.cb.2304002

Research Progress of Artificial Metalloenzymes

LIU Xiao-yan^**,HUANG Chao-qun^**,JIN Xue-rui,LUO Yun-zi^***(

)

Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

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Abstract

Enzymes with high efficiency and specificity have attracted much attention from researchers. Among them, metalloenzymes account for about 1/3 of natural enzymes. Metalloenzymes are generally composed of metal cofactors and corresponding protein scaffolds, in which the metal cofactors provide the active center. The protein scaffolds provide the chiral environment and attachment sites for metal cofactors. Existing studies have revealed that metalloenzymes fail to work without metal cofactors. The metal cofactors mainly exist in the form of metal ions or metal ligands. Among the natural metalloenzymes discovered so far, the metal elements in metal cofactors are mainly Fe, Cu, and Zn. Besides, there are also Mn and other metal elements. Metalloenzymes play an important role in organisms, including signal transduction and immune regulation. Various metalloenzymes can catalyze different reactions, such as hydroxylation and epoxidation. However, it is difficult for natural metalloenzymes to catalyze nonnatural substrates. Some metalloenzymes have low catalytic efficiency and poor stability in vitro, making them unable to be widely used. Recently, rapidly developed biotechnology has accelerated the development of metalloenzymes. By simulating natural metalloenzymes, artificial metalloenzymes (ArMs) have been constructed continuously. The appearance of ArMs has expanded reaction types. In summary, three main strategies have been applied in designing ArMs, including the reconstruction of cofactors, design of protein scaffolds, and modification of nanoparticles. The reconstruction of cofactors is mainly achieved by chemical modification and replacement. Design of protein scaffolds is achieved by selecting some stable structures and utilizing computer-aided methods. Notably, the development of nanotechnology has also provided good ideas for redesigning ArMs. The enzyme property can be improved by binding metalloenzymes to the surface of nanometers or being embedded in nanoparticles. Herein, we summarize some achievements of ArMs in recent years. A brief introduction about the challenges and opportunities faced by ArMs is provided, which is helpful for the design and application of ArMs.

Key words： Artificial metalloenzyme Cofactor Protein scaffold Rational design Nanotechnology

Received: 03 April 2023 Published: 02 November 2023

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Cite this article:

LIU Xiao-yan, HUANG Chao-qun, JIN Xue-rui, LUO Yun-zi. Research Progress of Artificial Metalloenzymes. China Biotechnology, 2023, 43(10): 72-84.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2304002 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I10/72

Fig.1 Strategies of artificial metalloenzymes construction Protein scaffolds design: Using de novo design to obtain scaffolds or using protein engineering to improve catalytic efficiency and develop new functions of artificial metalloenzymes. Cofactors reconstitution: Using chemical modification to improve the microenvironment of cofactors; introducing or replacing metal ligands to construct new artificial metalloenzymes. Enzymes modification assisted by nanotechnology: Encapsulating artificial metalloenzymes in nanoparticles or adsorbing on the surface to immobilize enzymes, allowing to increase stability and activity. The protein structure was from the Protein Data Bank (PDB) and edited by PyMOL

Fig.2 Artificial metalloenzymes construction via porphyrin substitution^[27] (a) The structure and active sites of CYP119 (PDB ID: 1IO7) (b) Enantioselectivity and yield of the conversion of diazoesters to dihydrobenzofuran catalyzed by evolved CYP119 variants (c) Extended reaction type catalyzed by CYP119 mutants, Ir(Me)-PIX CYP119-Max: L69V, T213G, C317G, V254L. The protein structure is from PDB and edited with PyMOL

蛋白骨架	原辅因子	新辅因子	功能	参考文献
Apo-SiRCcP.1	-	[4Fe-4S]	催化亚硫酸盐的还原反应	[11]
HSA	-	Au	催化氢胺化反应	[38]
Homo-oligomeric protein	-	Cu(bpy)	催化多质子/电子介导的氧化还原反应	[39]
MC6^*a	-	Fe、Mn	过加氧酶活性	[40]
αRepA3	-	Cu(II)	催化Diels-Alder反应	[41]
αRep	-	Co(III)-porphyrin complex	光诱导制氢和二氧化碳还原反应	[42]
MDRs	-	Cu(II)、BpyA_Cu(II)	催化Friedel-Crafts烷基化	[43]
mAbs	-	BIQ-Cu、BIQ-PdCl₂、BIQ-Pd(OAc)₂、 BIQ-PtCl₂	催化Friedel-Crafts烷基化反应	[44]
LmrR	-	Fe(III)-CPPIX	催化环丙烷化反应	[45]
LmrR	-	Cu(II)-phen complex	催化Friedel-Crafts烷基化反应	[46]
LmrR	-	Cu(II)-phenanthroline complex	催化Friedel- Crafts烷基化和 Diels - Alder反应	[47]
LmrR	-	Cu(II) complexes	催化迈克尔加成反应	[48]
Sav-SOD	-	Cp*Ir(biot-p-L)Cl	催化不对称氢转移反应	[49]
Nitrobindin (NB)	-	Cp*Rh(III) complexes	催化环加成反应	[50]
Nitrobindin (NB)	-	Cp*Rh(III)-dithiophosphate complex	催化环加成反应	[51]
POP	-	Dirhodium complexes	催化环丙烷化反应	[52]
POP	-	Ru(II) polypyridyl complexes	催化环加成反应	[53]
POP	-	Dirhodium complexes	催化重氮化偶合反应	[54]
(A3A3’)Y26C	-	Mn (III)-tetraphenylporphyrin	过氧化物酶和单加氧酶的活性	[55]
Four-helix bundle	-	Zn-PPIX	过氧化物酶活性	[56]
Four-helix bundle	-	Ru(II)(η6-arene)(bipyridine) complexes	催化氢转移反应	[57]
Van and DADA complexes	-	[IrCp*(m-I)Cl]Cl	催化环亚胺的不对称加氢反应	[58]
Heptapeptidic	-	Methyl salicylate Pd complexes	催化去炔丙基化和Suzuki - Miyaura 交叉偶联反应	[59]
TbADH	Zn (II)	Cp*Rh(III) complexes	催化还原反应	[24]
Azurin	Cu	Ni	催化碳碳耦合及硫酯合成反应	[60]
P450-BM3	Fe-PPIX	Ir(Me)-deuteroporphyrin IX	催化烯烃环丙烷化反应	[61]
CYP119	Fe-PPIX	Ir(Me)-PIX、Ir(Me)-MPIX	催化环丙烷化反应	[28,37,62]
Mb	Fe-PPIX	Fe-2, 4-diacetyl deuteroporphyrin IX	催化烯烃的不对称环丙烷化	[63]
Mb	Fe-PPIX	CuCP	DNA切割活性	[64]

Table 1 Summary of artificial metalloenzymes containing metal cofactors

Fig.3 Artificial metalloenzymes construction employing protein scaffold^[70] (a) Schematic diagram of ArM assembled by LmrR protein scaffold and copper cofactor (b) Friedel-Crafts alkylation of indoles catalyzed by ArM

Fig.4 De novo design strategy of protein scaffolds (a) Mechanism study of enzymatic reaction to find the key sites (b) Mining of stable protein structures and cofactors from databases (c) Rational design and assembly of the protein skeleton with cofactors (d) Screening of new artificial metalloenzymes. The protein structures are from PDB and edited with PyMOL

Fig.5 Strategies of artificial metalloenzymes modification assisted by nanotechnology (a) Immobilization of artificial metalloenzymes on the NPs surface using crosslinking agents (b) Immobilization of artificial metalloenzymes into the MOFs


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