人工Cu-TM1459金属酶催化不对称迈克尔加成反应

doi:10.11949/0438-1157.20240302

摘要/Abstract

摘要：

不对称迈克尔加成反应是合成手性化合物的重要反应，手性的构建一般由传统手性金属络合物催化完成，催化剂结构复杂，制备困难。人工金属酶可以利用生物大分子替代过渡金属手性催化剂，成为研究的热点。使用TM1459蛋白质支架，在原有的金属结合基序上理性引入两个组氨酸和一个羧酸盐面部三联基序，配位Cu²⁺，制备了人工Cu-TM1459金属酶。将其用于催化不对称迈克尔加成反应研究，Cu-H52A/H58E变体金属酶具有中等反应活性和较高对映选择性（e.e.值达58%）。进一步通过分子对接和催化机理研究对金属结合位点附近关键残基进行定点突变，I108A/C106V/K24E突变体催化该反应，产率99%，e.e.值93%。

关键词: 人工金属酶, 不对称迈克尔加成反应, 理性设计, 蛋白质, 生物催化, 催化剂

Abstract:

The asymmetric Michael addition reaction is an important reaction for synthesizing chiral compounds. Traditionally, chiral metal complexes are used as catalysts for chiral induction, but they are complex in structure and difficult to prepare. Artificial metalloenzymes, which utilize biomacromolecules to replace transition metal chiral catalysts, have become a research hotspot. In this study, two histidines and a carboxylate facial triple motif were rationally introduced on the original metal binding motif of TM1459 protein scaffold to coordinate Cu²⁺, and artificial Cu-TM1459 metalloenzyme was prepared. It was applied to catalyze the asymmetric Michael addition reaction, and the Cu-H52A/H58E variant metalloenzyme exhibited moderate reaction activity and high enantioselectivity (e.e. value up to 58%). Further studies on molecular docking and catalytic mechanism led to site-directed mutations of key residues near the metal binding site. The I108A/C106V/K24E mutant catalyzed the reaction with a yield of 99% and an e.e. value of 93%.

Key words: artificial metalloenzymes, asymmetric Michael addition reaction, rational design, protein, biocatalysis, catalyst

中图分类号:

TQ 028.8

张梦婷, 王书林, 桑熙, 元兴昊, 徐刚. 人工Cu-TM1459金属酶催化不对称迈克尔加成反应[J]. 化工学报, 2024, 75(9): 3255-3265.

Mengting ZHANG, Shulin WANG, Xi SANG, Xinghao YUAN, Gang XU. Artificial Cu-TM1459 metalloenzyme catalyzes asymmetric Michael addition reaction[J]. CIESC Journal, 2024, 75(9): 3255-3265.

图/表 15

图1 人工金属酶应用于不对称迈克尔加成反应

Fig.1 Artificial metalloenzymes applied to asymmetric Michael addition reactions

图2 fac-[M(His2)(OCOR)]三联体基序突变体库用于不对称迈克尔加成反应

Fig.2 fac-[M(His2)(OCOR)] triplet mutant library for asymmetric Michael addition reaction

表1 PCR反应体系

Table 1 Components of PCR system

试剂	体积/µl
PrimerStar^® MAX DNA聚合酶	25
DNA模板	0.5
正向引物	1
反向引物	1
dd H₂O	22.5

表2 PCR扩增步骤

Table 2 PCR steps

步骤	温度/℃	时间	循环次数
预变性	98	2.5 min	1
循环阶段	98	15 s	30
	55~65	15 s
	72	1.5 min
后延伸	72	5 min	1
保存	12	∞	—

图3 氮杂查尔酮类化合物1a和1b

Fig.3 Nitrogen-containing chalcone compounds 1a and 1b

表3 fac-［Cu（His2）（OCOR）］三联体突变体库筛选

Table 3 Screening of fac-［Cu（His2）（OCOR）］ trinuclear mutant library

序号	TM1459突变体	产率/%	e.e./%
1	—	12	—
2	Cu²⁺	28	—
3	apo-H52A/H58E	13	-14
4	H52A	31	8
5	H52A/H58E	45	58
6	H52A/H54E	40	-5
7	H52A/H92E	55	-12
8	H58A/H52E	49	-13
9	H58A/H54E	51	-30
10	H58A/H92E	43	-4

图4 人工Cu-TM1459金属酶催化反应体系优化

Fig.4 Optimization of catalytic reaction system of artificial Cu-TM1459 metalloenzyme

图5 配体1a的分子对接结果

Fig. 5 Molecular docking results of ligand 1a

图6 TM1459金属活性位点附近残基

Fig.6 Residues near active site of TM1459 metalloenzyme

图7 底物硝基甲烷与金属活性位点的相互作用

Fig.7 Interaction between substrate nitromethane and metal active sites

图8 人工Cu-TM1459金属酶催化不对称迈克尔加成反应的可能机理

Fig.8 Possible mechanism of asymmetric michael addition catalyzed by artificial Cu-TM1459 metalloenzyme

表4 关键氨基酸丙氨酸扫描

Table 4 Alanine scanning of key amino acids

进攻方向	突变体	产率/%	e.e./%
Re面	W56A	80	44
	I108A	96	93
Si面	K24A	75	31
	R39A	88	61
	I49A	88	-8
	F94A	67	19
Re面或Si面	C106A	80	62

图9 定点突变改善金属酶催化性能

Fig.9 Site-directed mutagenesis to enhance catalytic performance of metalloenzymes

Table 5 Optimizing catalytic performance of artificial Cu-TM1459 through combinatorial mutations

突变数/个	突变体	产率/%	e.e./%
2	I108A/C106V	79	92
3	I108A/C106V/W56Y	26	1
3	I108A/C106V/K24E	>99	93
4	I108A/C106V/K24E/W56Y	57	92

表5　组合突变优化人工Cu-TM1459的催化性能

参考文献 43

1	Rossiter B E, Swingle N M. Asymmetric conjugate addition[J]. Chemical Reviews, 1992, 92(5): 771-806.
2	Christoffers J, Koripelly G, Rosiak A, et al. Recent advances in metal-catalyzed asymmetric conjugate additions[J]. Synthesis, 2007, 2007(9): 1279-1300.
3	Rosati F, Roelfes G. Artificial metalloenzymes[J]. ChemCatChem, 2010, 2(8): 916-927.
4	Gennari C, Piarulli U. Combinatorial libraries of chiral ligands for enantioselective catalysis[J]. Chemical Reviews, 2003, 103(8): 3071-3100.
5	Schwizer F, Okamoto Y, Heinisch T, et al. Artificial metalloenzymes: reaction scope and optimization strategies[J]. Chemical Reviews, 2018, 118(1): 142-231.
6	Davis H J, Ward T R. Artificial metalloenzymes: challenges and opportunities[J]. ACS Central Science, 2019, 5(7): 1120-1136.
7	Steinreiber J, Ward T R. Artificial metalloenzymes as selective catalysts in aqueous media[J]. Coordination Chemistry Reviews, 2008, 252(5/6/7): 751-766.
8	Letondor C, Humbert N, Ward T R. Artificial metalloenzymes based on biotin-avidin technology for the enantioselective reduction of ketones by transfer hydrogenation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(13): 4683-4687.
9	Letondor C, Pordea A, Humbert N, et al. Artificial transfer hydrogenases based on the biotin-(strept)avidin technology: fine tuning the selectivity by saturation mutagenesis of the host protein[J]. Journal of the American Chemical Society, 2006, 128(25): 8320-8328.
10	Ward T R. Artificial metalloenzymes based on the biotin-avidin technology: enantioselective catalysis and beyond[J]. Accounts of Chemical Research, 2011, 44(1): 47-57.
11	Pordea A, Creus M, Letondor C, et al. Improving the enantioselectivity of artificial transfer hydrogenases based on the biotin-streptavidin technology by combinations of point mutations[J]. Inorganica Chimica Acta, 2010, 363(3): 601-604.
12	Roelfes G. DNA and RNA induced enantioselectivity in chemical synthesis[J]. Molecular BioSystems, 2007, 3(2): 126-135.
13	Coquière D, Bos J, Beld J, et al. Enantioselective artificial metalloenzymes based on a bovine pancreatic polypeptide scaffold [J]. Angewandte Chemie International Edition, 2009, 48(28): 5159-5162.
14	Coquière D, Feringa B L, Roelfes G. DNA-based catalytic enantioselective michael reactions in water[J]. Angewandte Chemie International Edition, 2007, 46(48): 9308-9311.
15	Bos J, García-Herraiz A, Roelfes G. An enantioselective artificial metallo-hydratase[J]. Chemical Science, 2013, 4(9): 3578-3582.
16	Sreenilayam G, Moore E J, Steck V, et al. Stereoselective olefin cyclopropanation under aerobic conditions with an artificial enzyme incorporating an iron-chlorin e6 cofactor[J]. ACS Catalysis, 2017, 7(11): 7629-7633.
17	Wang W J, Tachibana R, Zou Z, et al. Manganese transfer hydrogenases based on the biotin-streptavidin technology[J]. Angewandte Chemie International Edition, 2023, 62(43): e202311896.
18	Aplander K, Ding R, Krasavin M, et al. Asymmetric lewis acid catalysis in water: α-amino acids as effective ligands in aqueous biphasic catalytic Michael additions[J]. European Journal of Organic Chemistry, 2009, 2009(6): 810-821.
19	Dey S, Jäschke A. Tuning the stereoselectivity of a DNA-catalyzed Michael addition through covalent modification[J]. Angewandte Chemie International Edition, 2015, 54(38): 11279-11282.
20	Dong X C, Yuan Z J, Qu Y, et al. An ATP-Cu(Ⅱ) catalyst efficiently catalyzes enantioselective Michael reactions in water[J]. Green Chemistry, 2021, 23(24): 9876-9880.
21	Okrasa K, Kazlauskas R J. Manganese-substituted carbonic anhydrase as a new peroxidase[J]. Chemistry, 2006, 12(6): 1587-1596.
22	Fernández-Gacio A, Codina A, Fastrez J, et al. Transforming carbonic anhydrase into epoxide synthase by metal exchange[J]. Chembiochem, 2006, 7(7): 1013-1016.
23	Jing Q, Okrasa K, Kazlauskas R J. Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase: a new reductase[J]. Chemistry, 2009, 15(6): 1370-1376.
24	Markel U, Sauer D F, Schiffels J, et al. Towards the evolution of artificial metalloenzymes—a protein engineer’s perspective[J]. Angewandte Chemie International Edition, 2019, 58(14): 4454-4464.
25	Lewis J C. Beyond the second coordination sphere: engineering dirhodium artificial metalloenzymes to enable protein control of transition metal catalysis[J]. Accounts of Chemical Research, 2019, 52(3): 576-584.
26	Lesley S A, Kuhn P, Godzik A, et al. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline[J]. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(18): 11664-11669.
27	Silvennoinen L, Sandalova T, Schneider G. The polyketide cyclase RemF from Streptomyces resistomycificus contains an unusual octahedral zinc binding site[J]. FEBS Letters, 2009, 583(17): 2917-2921.
28	Roelfes G. LmrR: a privileged scaffold for artificial metalloenzymes[J]. Accounts of Chemical Research, 2019, 52(3): 545-556.
29	Matsumoto R, Yoshioka S, Yuasa M, et al. An artificial metallolyase with pliable 2-His-1-carboxylate facial triad for stereoselective Michael addition[J]. Chemical Science, 2023, 14(14): 3932-3937.
30	Fujieda N, Ichihashi H, Yuasa M, et al. Cupin variants as a macromolecular ligand library for stereoselective Michael addition of nitroalkanes[J]. Angewandte Chemie (International Ed. in English), 2020, 59(20): 7717-7720.
31	Fujieda N, Nakano T, Taniguchi Y, et al. A well-defined osmium-cupin complex: hyperstable artificial osmium peroxygenase[J]. Journal of the American Chemical Society, 2017, 139(14): 5149-5155.
32	Jaroszewski L, Schwarzenbacher R, von Delft F, et al. Crystal structure of a novel manganese-containing cupin (TM1459) from Thermotoga maritima at 1.65 Å resolution[J]. Proteins, 2004, 56(3): 611-614.
33	Woo E J, Dunwell J M, Goodenough P W, et al. Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities[J]. Nature Structural Biology, 2000, 7(11): 1036-1040.
34	Amrein B, Schmid M, Collet G, et al. Identification of two-histidines one-carboxylate binding motifs in proteins amenable to facial coordination to metals[J]. Metallomics: Integrated Biometal Science, 2012, 4(4): 379-388.
35	Bruijnincx P C A, van Koten G, Klein Gebbink R J M. Mononuclear non-heme iron enzymes with the 2-His-1-carboxylate facial triad: recent developments in enzymology and modeling studies[J]. Chemical Society Reviews, 2008, 37(12): 2716-2744.
36	Koehntop K D, Emerson J P, Que L. The 2-His-1-carboxylate facial triad: a versatile platform for dioxygen activation by mononuclear non-heme iron(Ⅱ) enzymes[J]. Journal of Biological Inorganic Chemistry, 2005, 10(2): 87-93.
37	Parkin G. Synthetic analogues relevant to the structure and function of zinc enzymes[J]. Chemical Reviews, 2004, 104(2): 699-767.
38	Podtetenieff J, Taglieber A, Bill E, et al. An artificial metalloenzyme: creation of a designed copper binding site in a thermostable protein[J]. Angewandte Chemie International Edition, 2010, 49(30): 5151-5155.
39	Fink M, Trunk S, Hall M, et al. Engineering of TM1459 from Thermotoga maritima for increased oxidative alkene cleavage activity[J]. Frontiers in Microbiology, 2016, 7: 1511.
40	Grill B, Pavkov-Keller T, Grininger C, et al. Engineering TM1459 for stabilisation against inactivation by amino acid oxidation[J]. Chemie Ingenieur Technik, 2023, 95(4): 596-606.
41	Otto S, Bertoncin F, Engberts J. Lewis acid catalysis of a Diels-Alder reaction in water[J]. Journal of the American Chemical Society, 1996, 118: 7702-7707.
42	Grimm A R, Sauer D F, Davari M D, et al. Cavity size engineering of a β-barrel protein generates efficient biohybrid catalysts for olefin metathesis[J]. ACS Catalysis, 2018, 8(4): 3358-3364.
43	Reetz M T, Rentzsch M, Pletsch A, et al. A robust protein host for anchoring chelating ligands and organocatalysts[J]. Chembiochem, 2008, 9(4): 552-564.

编辑推荐

Metrics

阅读次数

全文

147

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
2	0	20	14	0	111

来源	本网站	其他网站

次数	78	69
比例	53%	47%

摘要

[1]	王冉, 王焕, 熊晓云, 关慧敏, 郑云锋, 陈彩琳, 秦玉才, 宋丽娟. FCC催化剂传质强化活性位利用效率的可视化分析[J]. 化工学报, 2024, 75(9): 3198-3209.
[2]	刘亚超, 谭晓杰, 李旭东, 王瑞, 王慧, 韩璇, 赵青山. DES合成高活性CoCO₃纳米片及析氧反应性能研究[J]. 化工学报, 2024, 75(9): 3320-3328.
[3]	王树振, 王玉婷, 马梦茜, 张巍, 向江南, 鲁海莹, 王琰, 范彬彬, 郑家军, 代卫炯, 李瑞丰. 两步晶化合成ZSM-22分子筛及其临氢异构反应性能[J]. 化工学报, 2024, 75(9): 3176-3187.
[4]	罗莉, 陈文尧, 张晶, 钱刚, 周兴贵, 段学志. 氧化铝结构与表面性质调控及其催化甲醇脱水制二甲醚性能研究[J]. 化工学报, 2024, 75(7): 2522-2532.
[5]	吴哲明, 张碧云, 郑仁朝. 腈水解酶立体选择性改造及其合成布瓦西坦[J]. 化工学报, 2024, 75(7): 2633-2643.
[6]	刘旭升, 李泽洋, 杨宇森, 卫敏. 电催化二氧化碳还原制备气态产物的研究进展[J]. 化工学报, 2024, 75(7): 2385-2408.
[7]	王寅, 初鹏飞, 刘虎, 吕静, 黄守莹, 王胜平, 马新宾. 不同pH铝溶胶对二甲醚羰基化成型丝光沸石催化剂性能的影响[J]. 化工学报, 2024, 75(7): 2533-2543.
[8]	杨露, 刘聪聪, 孟彤彤, 张博远, 杨腾飞, 邓文安, 王晓斌. 分散型催化剂在煤/重油共炼体系中的加氢抑焦作用[J]. 化工学报, 2024, 75(7): 2556-2564.
[9]	王天闻, 闫肃, 赵梦园, 杨天让, 刘建国. 固体氧化物电池空气电极铬中毒机理及抗铬性能研究进展[J]. 化工学报, 2024, 75(6): 2091-2108.
[10]	丁禹, 杨昌泽, 李军, 孙会东, 商辉. 原子尺度钼系加氢脱硫催化剂的研究进展与展望[J]. 化工学报, 2024, 75(5): 1735-1749.
[11]	赵亭亭, 鄢立祥, 唐福利, 肖敏之, 谭烨, 宋刘斌, 肖忠良, 李灵均. 光辅助锂-二氧化碳电池催化剂的设计策略与反应机理研究进展[J]. 化工学报, 2024, 75(5): 1750-1764.
[12]	莫锦洪, 韩雪, 朱毅翔, 李菁, 王旭裕, 纪红兵. Pt-Ga/CeO₂-ZrO₂-Al₂O₃脱氢裂解双功能催化剂用于正丁烷催化制烯烃研究[J]. 化工学报, 2024, 75(5): 1855-1869.
[13]	范以薇, 刘威, 李盈盈, 王培霞, 张吉松. 有机液体储氢中全氢化乙基咔唑催化脱氢研究进展[J]. 化工学报, 2024, 75(4): 1198-1208.
[14]	贾旭东, 杨博龙, 程前, 李雪丽, 向中华. 分步负载金属法制备铁钴双金属位点高效氧还原电催化剂[J]. 化工学报, 2024, 75(4): 1578-1593.
[15]	严孝清, 赵瑛, 张宇哲, 欧鸿辉, 黄起中, 胡华贵, 杨贵东. 五重孪晶铜纳米线@聚吡咯制备及其电催化硝酸盐还原制氨[J]. 化工学报, 2024, 75(4): 1519-1532.