Jin Wang, Han Ke, Jun Yang, Nianxin Guo, Kangdelong Hu, Ruyao Tang, Qi Ding, Lei Gao,*and Xiaoguang Lei*
Chem Catalysis 3, 1–12, January 19, 2023
https://doi.org/10.1016/j.checat.2022.10.027
Substituted cyclohexanes are common scaffolds found in both natural products and drug molecules. Diels-Alderases that can efficiently catalyze intermolecular Diels-Alder reactions to generate cyclohexene ring systems have received considerable interest. However, the synthetic power of Diels-Alderases is incomparable with chemo-catalysts due to their limited substrate scopes. Here, we report a new chemo-enzymatic strategy for the diversity-oriented syntheses of functionalized cyclohexenes. We first applied focused rational iterative site-specific mutagenesis to generate a natural Diels-Alderase variant M3, which shows a 34-fold increase in catalytic efficiency, broad substrate scope, and good to perfect stereoselectivity.Then, we used diverse transition-metal-catalyzed decarboxylative coupling reactions to functionalize the enzymatic Diels-Alder products.This work offers an efficient synthetic route to structurally diverse cyclohexenes that are not accessible by solely using biocatalysis or chemo-catalysis and illustrates how chemo-catalysis can cooperate with biocatalysis to expand the synthetic application of biocatalysts.
Special Issue: Microbiome Received: May 8, 2022 Revised: September 2, 2022
Bile acids are essential metabolites and signaling molecules in mammals. Primary bile acids are synthesized from cholesterol in the liver. At the same time, the microbiota in the mammalian gut has many interactions with bile acid, including various biotransformation processes such as 7-dehydroxylation and 3- epimerization. 7-Dehydroxylation is mediated by a bile acid-inducible (bai) operon, while 7-dehydroxylation and 3-epimerization are independently observed in only a few strains. Herein, we describe a novel microbe, Dorea sp. AM58-8, that can accomplish a two-steptransformation and turn primary bile acids into both 3α secondary bile acids like deoxycholic acid and lithocholic acid, and 3β secondary bile acids like isodeoxycholic acid and isolithocholic acid. We subsequently characterized BaiA, BaiB, BaiE, and their substrate profiles biochemically. The potential bai gene clusters in the metagenomes were further mined. Their evolution, potential functions, and possible regulatory pathways were predicted using bioinformatics based on our understanding of the 7-dehydroxylation pathway in Dorea sp. AM58-8. This study of Dorea sp. AM58-8 also helps us distinguish the inactive bacteria that seem to have the 7- dehydroxylation pathway proteins and discover the 7-dehydroxylation pathway in other mammalian gut microbes.
Lei Gao, Jun Yang, Xiaoguang Lei* Tetrahedron Chem 2022, 2, 10001
The Diels-Alder (D-A) reaction is one of the most critical chemical transformations to construct C–C bonds with predictable regio- and stereo-selectivities. It has been widely used as a retrosynthetic disconnection in synthetic chemistry. Although significant advances have been made, synthetic challenges remain in how to precisely control the stereochemistry of intermolecular Diels-Alder reaction. Enzymes are well known for their remarkable catalytic efficiency and selectivity compared with chemo-catalysts. Therefore, identifying and designing intermolecular Diels-Alderases that can be used in organic synthesis have received considerable attention from the synthetic community. In this review, we review all the enzymes capable of catalyzing formal intermolecular Diels-Alder reactions in natural product biosynthetic pathways, discuss their catalytic mechanisms in detail, and highlight their synthetic potential in the precise and efficient synthesis of enantiopure D-A products. We also discuss the different strategies that can be used to create new artificial Diels-Alderases, especially RNA-based Diels-Alderases
Junping Fan, Linghan Hu, Zongwei Yue, Daohong Liao, Fusheng Guo, Han Ke, Daohua Jiang, Yong Yang & Xiaoguang Lei Nature Chemical Biology (2023) https://doi.org/10.1038/s41589-022-01166-5
The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.
Publication Date:October 20, 2022 https://doi.org/10.1021/acs.analchem.2c02116 Subcellular protein−protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical crosslinking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.
Bile acids (BAs) are a class of endogenous metabolites with important functions. As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive the BA stress and knowledge is limited about how they develop BA tolerance. In this work, we applied a quantitative chemoproteomic strategy to profile BAinteracting proteins in bacteria, aiming to discover the sensing pathway of BAs. Using a clickable and photo-affinity BA probe with quantitative mass spectrometry, we identified a list of histidine kinases (HKs) of the twocomponent systems (TCS) in bacteria as the novel binding targets of BA. Genetic screening revealed that knocking out one specific HK, EnvZ, renders bacteria with significant sensitivity to BA. Further biochemical and genetic experiments demonstrated that BA binds to a specific pocket in EnvZ and activates a downstream signaling pathway to help efflux of BA from bacteria, resulting in BA tolerance. Collectively, our data revealed that EnvZ is a novel sensor of BA in bacteria and its associated TCS signaling pathway plays a critical role in mediating bacterial BA tolerance, which opens new opportunities to combat BA-tolerating pathogens.
The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process.
Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart.
Jianyong Du, Lixia Zheng,Peng Gao, Hang Yang, Wan-Jie Yang, Fusheng Guo,Ruqi Liang,Mengying Feng, Zihao Wang,Zongwang Zhang, Linlu Bai,Ye Bu, Shijia Xing, Wen Zheng, Xuelian Wang, Li Quan, Xinli Hu, Haosen Wu,Zhixing Chen, Liangyi Chen, Ke Wei, Zhe Zhang, Xiaojun Zhu, Xiaolin Zhang, Qiang Tu, Shi-Min Zhao,* Xiaoguang Lei,* and Jing-Wei Xiong.*
Cell Stem Cell 2022, 29, 545.
Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart
Jun Zhang, Daohong Liao, Rongchang Chen, Fangfang Zhu, Yaqing Ma, Lei Gao,Ge Qu, Chengsen Cui, Zhoutong Sun,* Xiaoguang Lei,* and Shu-Shan Gao* Angew. Chem. Int. Ed. 2022, 61, e202201908.
Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm, and a significant increase in the e.e. value from 78% (R) to >99% (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.
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Diversity-oriented synthesis of cyclohexenes by combining enzymatic intermolecular Diels- Alder reactions and decarboxylative functionalizations
Jin Wang, Han Ke, Jun Yang, Nianxin Guo, Kangdelong Hu, Ruyao Tang, Qi Ding, Lei Gao,*and Xiaoguang Lei*
Chem Catalysis 3, 1–12, January 19, 2023
https://doi.org/10.1016/j.checat.2022.10.027
Substituted cyclohexanes are common scaffolds found in both natural products and drug molecules. Diels-Alderases that can efficiently catalyze intermolecular Diels-Alder reactions to generate cyclohexene ring systems have received considerable interest. However, the synthetic power of Diels-Alderases is incomparable with chemo-catalysts due to their limited substrate scopes. Here, we report a new chemo-enzymatic strategy for the diversity-oriented syntheses of functionalized cyclohexenes. We first applied focused rational iterative site-specific mutagenesis to generate a natural Diels-Alderase variant M3, which shows a 34-fold increase in catalytic efficiency,
broad substrate scope, and good to perfect stereoselectivity.Then, we used diverse transition-metal-catalyzed decarboxylative coupling reactions to functionalize the enzymatic Diels-Alder products.This work offers an efficient synthetic route to structurally diverse cyclohexenes that are not accessible by solely using biocatalysis or chemo-catalysis and illustrates how chemo-catalysis can cooperate with biocatalysis to expand the synthetic application
of biocatalysts.
A Novel Gene Alignment in Dorea sp. AM58‑8 Produces 7‑Dehydroxy-3β Bile Acids from Primary Bile Acids
Yingjie Bai, Tianhu Zhao, Mengyu Gao, Yuanqiang Zou, and Xiaoguang Lei*
https://doi.org/10.1021/acs.biochem.2c00264
Special Issue: Microbiome
Received: May 8, 2022
Revised: September 2, 2022
Bile acids are essential metabolites and signaling molecules in mammals. Primary bile acids are synthesized from cholesterol in the liver. At the same time, the microbiota in the mammalian gut has many interactions with bile acid, including various biotransformation processes such as 7-dehydroxylation and 3- epimerization. 7-Dehydroxylation is mediated by a bile acid-inducible (bai) operon, while 7-dehydroxylation and 3-epimerization are independently observed in only a few strains. Herein, we describe a novel microbe, Dorea sp. AM58-8, that can accomplish a two-steptransformation and turn primary bile acids into both 3α secondary bile acids like deoxycholic acid and lithocholic acid, and 3β secondary bile acids like isodeoxycholic acid and isolithocholic acid. We subsequently characterized BaiA, BaiB, BaiE, and their substrate profiles biochemically. The potential bai gene clusters in the metagenomes were further mined. Their evolution, potential functions, and possible regulatory pathways were predicted using bioinformatics based on our understanding of the 7-dehydroxylation pathway in Dorea sp. AM58-8. This study of Dorea sp. AM58-8 also helps us distinguish the inactive bacteria that seem to have the 7- dehydroxylation pathway proteins and discover the 7-dehydroxylation pathway in other mammalian gut microbes.
Enzymatic intermolecular Diels-Alder reactions in synthesis: From nature to design
Lei Gao, Jun Yang, Xiaoguang Lei* Tetrahedron Chem 2022, 2, 10001
The Diels-Alder (D-A) reaction is one of the most critical chemical transformations to construct C–C bonds with predictable regio- and stereo-selectivities. It has been widely used as a retrosynthetic disconnection in synthetic chemistry. Although significant advances have been made, synthetic challenges remain in how to precisely control the stereochemistry of intermolecular Diels-Alder reaction. Enzymes are well known for their remarkable catalytic efficiency and selectivity compared with chemo-catalysts. Therefore, identifying and designing intermolecular Diels-Alderases that can be used in organic synthesis have received considerable attention from the synthetic community. In this review, we review all the enzymes capable of catalyzing formal intermolecular Diels-Alder reactions in natural product biosynthetic pathways, discuss their catalytic mechanisms in detail, and highlight their synthetic potential in the precise and efficient synthesis of enantiopure D-A products. We also discuss the different strategies that can be used to create new artificial Diels-Alderases, especially RNA-based Diels-Alderases
Structural basis of TRPV3 inhibition by an antagonist
Junping Fan, Linghan Hu, Zongwei Yue, Daohong Liao, Fusheng Guo, Han Ke, Daohua Jiang, Yong Yang & Xiaoguang Lei
Nature Chemical Biology (2023) https://doi.org/10.1038/s41589-022-01166-5
The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.
Subcellular Interactomes Revealed by Merging APEX with Cross- Linking Mass Spectrometry
Mengze Sun, Feng Yuan, Yuliang Tang, Peng Zou,* and Xiaoguang Lei*
Anal. Chem. 2022, 94, 14878-14888.
Publication Date:October 20, 2022
https://doi.org/10.1021/acs.analchem.2c02116
Subcellular protein−protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical crosslinking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.
Chemoproteomic Profiling Reveals the Mechanism of Bile Acid Tolerance in Bacteria
Biwei Liu, Shentian Zhuang, Runze Tian, Yuan Liu, Yanqi Wang, Xiaoguang Lei,* and Chu Wang* ACS Chemical Biology Article ASAP
DOI: 10.1021/acschembio.2c00286
Bile acids (BAs) are a class of endogenous metabolites with important functions. As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive the BA stress and knowledge is limited about how they develop BA tolerance. In this work, we applied a quantitative chemoproteomic strategy to profile BAinteracting proteins in bacteria, aiming to discover the sensing pathway of BAs. Using a clickable and photo-affinity BA probe with quantitative mass spectrometry, we identified a list of histidine kinases (HKs) of the twocomponent systems (TCS) in bacteria as the novel binding targets of BA. Genetic screening revealed that knocking out one specific HK, EnvZ, renders bacteria with significant sensitivity to BA. Further biochemical and genetic
experiments demonstrated that BA binds to a specific pocket in EnvZ and activates a downstream signaling pathway to help efflux of BA from bacteria, resulting in BA tolerance. Collectively, our data revealed that EnvZ is a novel sensor of BA in bacteria and its associated TCS signaling pathway plays a critical role in mediating bacterial BA tolerance, which opens new opportunities to combat BA-tolerating pathogens.
Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry
Tiantian Wei, Jue Wang, Ruqi Liang, Wendong Chen, Yilan Chen, Mingzhe Ma, An He, Yifei Du, Wenjing Zhou, Zhiying Zhang, Xin Zeng, Chu Wang, Jin Lu, Xing Guo, Xiao-Wei Chen, Youjun Wang, Ruijun Tian, Junyu Xiao, Xiaoguang Lei
eLife 2022, 11 :e77696. https://doi.org/10.7554/eLife.77696
The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process.
Hesperadin Is a Novel CaMKII-δ Inhibitor Hesperadin Exerts Dual Functions to Ameliorate Cardiac Ischemia/Reperfusion Injury and Inhibit Tumor Growth
Junxia Zhang; Ruqi Liang; Kai Wang; Wenjia Zhang; Mao Zhang; Li Jin; Peng Xie; Wen Zheng; Haibao Shang; Qingmei Hu; Jiayi Li; Gengjia Chen; Fujian Wu; Feng Lan; Lipeng Wang; Shi-Qiang Wang; Yongfeng Li; Yong Zhang; Jinghao Liu; Fengxiang Lv; Xinli Hu; Rui-Ping Xiao; Xiaoguang Lei; Yan Zhang.
Circulation 2022, 145, 1154-1168
Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease, the leading cause of morbidity and mortality worldwide. At present, there is no effective therapy for reducing cardiac I/R injury. CaMKII (Ca2+/calmodulin-dependent kinase II) plays a pivotal role in the pathogenesis of severe heart conditions, including I/R injury. Pharmacological inhibition of CaMKII is an important strategy in the protection against myocardial damage and cardiac diseases. To date, there is no drug targeting CaMKII for the clinical therapy of heart disease. Furthermore, at present, there is no selective inhibitor of CaMKII-δ, the major CaMKII isoform in the heart.
A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration
Jianyong Du, Lixia Zheng,Peng Gao, Hang Yang, Wan-Jie Yang, Fusheng Guo,Ruqi Liang,Mengying Feng, Zihao Wang,Zongwang Zhang, Linlu Bai,Ye Bu, Shijia Xing, Wen Zheng, Xuelian Wang, Li Quan, Xinli Hu, Haosen Wu,Zhixing Chen, Liangyi Chen, Ke Wei, Zhe Zhang, Xiaojun Zhu, Xiaolin Zhang, Qiang Tu, Shi-Min Zhao,* Xiaoguang Lei,* and Jing-Wei Xiong.*
Cell Stem Cell 2022, 29, 545.
Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart
Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering
Jun Zhang, Daohong Liao, Rongchang Chen, Fangfang Zhu, Yaqing Ma, Lei Gao,Ge Qu, Chengsen Cui, Zhoutong Sun,* Xiaoguang Lei,* and Shu-Shan Gao*
Angew. Chem. Int. Ed. 2022, 61, e202201908.
Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm, and a significant increase in the e.e. value from 78% (R) to >99% (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.