Isothiocyanates (ITCs) are a unique class of electrophilic natural products that exert biological effects by reacting with proteinous cysteines to generate thionoacyl adducts. However, the identification of ITCs’ target sites is still an unmet task due to the high lability of such adducts. Here, we report an unexpected chemistry through which the ITC-protein adduct forms a stable N-terminal dihydrothiazole peptide adduct during proteolysis. This proteolysis-assisted cyclization (PAC) reaction can be harnessed for developing affinity-based and activity-based chemoproteomic methods to site-specifically profile targets of ITCs. Applying these methods not only adds further complexity to the known polypharmacological landscape of sulforaphane but also expands the ligandable cysteinome with site-level resolution through a 55-member ITC library. Given the promising chemopreventive and therapeutic effects of ITCs, the PAC-based chemoproteomic platform may lay the groundwork for elucidating their mechanisms of action and ultimately diversifying cysteine targetability for drug discovery.
Zhanli Wang, Lu Han, Lei Gao*, Liyun Zhang, Yan Xie, Hanwu Liu, Junping Fan, Ming Wu, Ning Yue, Yan Wang, Meng Han, Tongcan Sun, Qi Ding, Xiyin Zheng, Jidong Cao, Xueqi Shen, Haijun Wang, Tuxunaili Aizitili, Chunyan Wu, Xuehong Wu, Zhenhua Liu, Yiguo Hong, Xiaoguang Lei*, and Yule Liu*
Cell, Available online 8 May 2026
https://doi.org/10.1016/j.cell.2026.04.021
Phytoalexins are core components of plant chemical defense against pathogens. However, the genetic basis and regulatory mechanisms governing their biosynthesis remain preliminary. Debneyol is a well-defined, broad-spectrum fungicidal phytoalexin. Here, we elucidate its biosynthetic pathway, key regulators, and activity against multiple pathogens. We show that debneyol is synthesized from farnesyl pyrophosphate (FPP) through three steps catalyzed by 5-epi-aristolochene synthase (EAS), 5-epi-aristolochene epoxidase (EAE), and epoxide hydrolase-1 (EH1). MCD1 (miR1919-targeted cell death-factor-1) interacts with EAS and EAE, enhancing their association and EAE activity and promoting debneyol biosynthesis. Increased MCD1expression confers plant resistance not only against fungal but also viral and bacterial pathogens. Our work reveals a complete plant phytoalexin-based chemical defense machinery, opening avenues for engineering broad-spectrum plant resistance and industrial-scale debneyol production via synthetic biology.
Jun Yang#, Ruichao Shen#, Chunyu Wang#, Wenneng Zhu, Han Ke, Junping Fan, Mengna Zhang, Yingjun Liu, Shuai Li, Guochuan Li, Xiaoming Wang, Yulong Li, Can Cao*, Xiaoguang Lei*
Nature Chemical Biology (2026)
https://doi.org/10.1038/s41589-026-02195-0
Chronic itch, particularly in cholestatic and uremic conditions, poses a notable clinical burden, yet treatment options remain inadequate. MRGPRX4 (hX4), a bile-acid-sensing G-protein-coupled receptor predominantly expressed in human sensory neurons, has emerged as a critical mediator of cholestatic pruritus. Here we identified and characterized HEP-50768, a potent and selective small-molecule inverse agonist of hX4 through high-throughput screening and structure–activity optimization. Structural elucidation through cryo-electron microscopy of the hX4–inverse agonist complex structure revealed the unique binding mode and inhibitory mechanism of HEP-50768. In hX4-humanized rats, HEP-50768 robustly suppressed bile-acid-induced pruritic behaviors. Comprehensive preclinical absorption, distribution, metabolism, excretion and safety profiling was performed in both rats and monkeys, and these findings establish HEP-50768 as a promising therapeutic candidate for chronic itch, supporting its advancement to clinical evaluation.
Glucosinolates (GLSs) play crucial roles in plant defense against herbivores. GTR1 facilitates the high-affinity transport of GLSs through a proton-dependent process. However, the molecular mechanism underlying GLS recognition and transport by GTR1 remains largely unknown. Here, we present four cryo-EM structures of Arabidopsis GTR1 in distinct states, namely, the outward-apo, inward-apo, 4MTB-bound and 3IMG-bound forms, revealing the structural basis for GLS and proton cotransport by GTR1. GTR1 consists of an MFS-like transmembrane domain and an intracellular domain (ICD). The ICD plays an essential role in GTR1 function by interacting with the gating helix, transmembrane helix 7. GLSs are recognized by the central cavity residues and directly interact with the conserved E1X1X2E2K motif. Our structural and functional analyses demonstrated that the E1X1X2E2K motif and Glu513 determine the proton coupling of GTR1. This study provides mechanistic insights into how GTR1 transports GLSs, which could aid in improving crop quality and enhancing resistance to herbivory.
Lei Gao#, Xiang Qiu#, Jun Yang#, Kangdelong Hu#, Peilin Li, Wei Li, Feng Gao, Fabrice Gallou, Florian Kleinbeck, Xiaoguang Lei*
Science 391,eadw3365 (2026)
https://doi.org/10.1126/science.adw336
Amide bond formation is widely used in pharmaceutical synthesis, typically involving stoichiometric coupling reagents to activate carboxylic acid substrates for a condensation reaction. As an alternative approach, we repurposed aldehyde dehydrogenases into oxidative amidases by creating a more hydrophobic and spacious catalytic pocket for amines to capture the thioester intermediate. This biocatalyst efficiently facilitates the formation of amide bonds between diverse aldehydes and amines. We also developed a two-step enzymatic cascade to synthesize amides from broadly available aliphatic alcohols. This biocatalytic strategy enabled the redesign of synthetic routes for five drug molecules. Our findings highlight the potential of oxidative amidases in advancing the synthesis of structurally diverse drug molecules through efficient amide bond formation.
Ke Wang, Junping Fan, Huiwen Chen, Bo Huang, Cheng Chi, Rui Yan, Di Wu, Feng Zhou, Wenhua Zhang, Juquan Jiang, Xiaoguang Lei & Daohua Jiang
Nature 651, 251–259 (2026)
https://doi.org/10.1038/s41586-025-09934-8
Bile acids (BAs) are crucial amphipathic surfactants that function as multifaceted regulators in various physiological processes, including nutrient absorption and distribution, lipid metabolism and inflammation. The human organic solute transporter αβ (OSTα–OSTβ; hereafter referred to as OSTα/β) is a BA transporter that has a key role in the secretion and distribution of BAs. Pathogenic mutations in OSTα/β have been associated with cholestasis. Despite the functional importance of OSTα/β in BA homeostasis, the stoichiometry and assembly of the complex and the molecular mechanism that underlies BA transport by OSTα/β remain unknown. Here we present cryo-electron microscopy structures of human OSTα/β in complex with cholesterols and an endogenous substrate, elucidating the structural basis for the function of OSTα/β. OSTα/β is assembled in a novel dimer-of-heterodimers manner: two OSTα units form the homodimeric core, with two OSTβ units bound to the periphery. OSTα adopts the G-protein-coupled-receptor (GPCR) fold and contains a unique cysteine-rich loop with seven palmitoylation sites; these cooperate with transmembrane helices 5 and 6, constituting a BA recognition site. A positive cavity in OSTα connects the BA site and facilitates the transmembrane translocation of BAs through OSTα/β. Together, this study reveals the architecture and transport mechanism of OSTα/β and provides insights into the structure–function relationships of this crucial transporter in BA homeostasis.
Cancer immunotherapies have revolutionized cancer treatment, yet many patients fail to respond. Activating innate immunity offers a promising approach to enhance therapeutic efficacy, but the signaling kinases directly regulating this process to boost antitumor responses remain elusive. Here we conduct an in vivo kinome CRISPR screen and identify CDK10 as a key suppressor of tumor immune surveillance. Mechanistically, CDK10 phosphorylates DNMT1 and RAP80 to reduce the accumulation of double-stranded RNA and R-loops, which alleviates the activation of innate immune pathways mediated by MDA5 and cGAS. Kinase inhibitor screens identify NVP-AST487 and ponatinib as selective CDK10 inhibitors. Both genetic and pharmacological inhibition of CDK10 activates MDA5 and cGAS pathways, fostering an immunoactive tumor microenvironment that enhances cancer immunotherapy in multiple mouse tumor models. Clinically, low CDK10 expression in tumors correlates with better immunotherapy responses. These findings establish CDK10 as a pivotal modulator of tumor immunity and a potential therapeutic target.
Mengze Sun , Baiyan Lu, Ying Yang, Junping Fan, Weiwei Ren, Xiaonan Chu, Yihui Gao, Jun Wu, Jue Wang, Han Ke, Zhiwen Liu, Shaojun Dai, Xiaoguang Lei*, and Chao Li*
PNAS 122(45), e2515322122 (2025)
https://doi.org/10.1073/pnas.2515322122
Since its initial identification as the receptor for Rapid Alkalinization Factor 1 (RALF1), FERONIA (FER) receptor kinase has emerged as a central signaling hub coordi nating plant development, stress adaptation, and immune responses. Nevertheless, fundamental questions persist regarding the precise mechanisms of FER-mediated signal transduction and its context-dependent functional specialization in multicel lular processes. Here, we develop Ferovicin (FRV), a small-molecule inhibitor that specifically disrupts FER kinase activity, thereby enabling mechanistic dissection of FER. Cocrystallization and mutational analysis show that FRV selectively binds to the ATP-binding pocket of the kinase domain of FER and inhibits its kinase activity. Assisted by the FRV tool and quantitative phosphoproteomics, we characterized a series of signaling pathways and networks regulated by RALF1 and FER. Notably, our analysis reveals that RALF1 activates FER through phosphorylation at Ser695, which subsequently inhibits H+ -ATPase1/2 via phosphorylation at Ser899. This mechanism leads to apoplastic alkalinization and regulates cell expansion in the root meristem. Given the conservation of FRV binding sites in FER proteins across land plant species, FRV will serve as a valuable tool for dissecting FER signaling mechanisms as well as facilitating agricultural applications.
Arginine, a critical amino acid for protein structure and function, is involved in enzyme catalysis and macromolecular interactions. However, selectively targeting its reactive guanidine group has been challenging. Here, we utilized a probe, AP-1, based on phenylglyoxal, which demonstrated remarkable chemical selectivity and reactivity toward arginine residues. Using activity-based protein profifiling (ABPP), we explored the human proteome across four cancer cell lines, obtaining quantitative data for approximately 17 000 arginine residues. This analysis led to the identifification of several previously unreported hyperreactive arginine residues, including R43 of PKM, R171 of LDHA, R172 of LDHB, R341 of CKB, R168 of EIF4A1, and R118 of FUBP1, which are crucial for protein function. Notably, the mutation of CKB’s R341 inhibited cell proliferation and migration by downregulating energy supply. We also introduced ArGO-LDHA-1, a covalent inhibitor targeting LDHA’s hyperreactive arginine residues, showing potential to enhance chemotherapy effificacy. This work highlights the biological signifificance of arginine residues and provides a platform for large-scale profifiling of arginine reactivity.
Nianxin Guo, Jun Gu, Qingyang Zhou, Fang Liu, Haoran Dong, Qi Ding, Qixuan Wang, Dongshan Wu, Jun Yang, Junping Fan, Lei Gao*, Kendall N. Houk*, and Xiaoguang Lei*
PNAS 122 (34), e2504346122 (2025)
https://doi.org/10.1073/pnas.2504346122
Berberine bridge enzyme (BBE)-like enzymes catalyze various oxidative cyclization and dehydrogenation reactions in natural product biosynthesis, but the molecular mechanism underlying the selectivity remains unknown. Here, we elucidated the catalytic mechanism of BBE-like oxidases from Morus alba involved in the oxidative cyclization and dehydrogenation of moracin C. X-ray crystal structures of a functionally promiscuous flavin adenine dinucleotide (FAD)–bound oxidase, MaDS1, with and without an oxidative dehydrogenation product were determined at 2.03 Å and 2.21 Å resolution, respectively. Structure-guided mutagenesis and sequence analysis have identified a conserved aspartic acid that directs the reaction toward the oxidative dehydrogenation pathway. A combination of density functional theory (DFT) calculations and molecular dynamics (MD) simulations has revealed that aspartic acid acts as the catalytic base to deprotonate the carbon-cation intermediate to generate the dehydrogenated product, which otherwise undergoes a spontaneous 6π electrocyclization in the oxidative cyclization pathway to furnish the 2H-benzopyran product.
-
-
-
-
-
-
-
-
-
-
Proteolysis-Assisted Cyclization Facilitates Site-Centric Target Deconvolution of Isothiocyanates
Jingyang He, Caiping Tian, Qiang Li, Jian Zhang, Jixiang He, Lingqiang Zhang, Xiaoguang Lei, Jing Yang
Angew. Chem. Int. Ed. 2026, e5512891
http://doi.org/10.1002/anie.5512891
Isothiocyanates (ITCs) are a unique class of electrophilic natural products that exert biological effects by reacting with proteinous cysteines to generate thionoacyl adducts. However, the identification of ITCs’ target sites is still an unmet task due to the high lability of such adducts. Here, we report an unexpected chemistry through which the ITC-protein adduct forms a stable N-terminal dihydrothiazole peptide adduct during proteolysis. This proteolysis-assisted cyclization (PAC) reaction can be harnessed for developing affinity-based and activity-based chemoproteomic methods to site-specifically profile targets of ITCs. Applying these methods not only adds further complexity to the known polypharmacological landscape of sulforaphane but also expands the ligandable cysteinome with site-level resolution through a 55-member ITC library. Given the promising chemopreventive and therapeutic effects of ITCs, the PAC-based chemoproteomic platform may lay the groundwork for elucidating their mechanisms of action and ultimately diversifying cysteine targetability for drug discovery.
Genetic basis of phytoalexin-mediated chemical defense in plants
Zhanli Wang, Lu Han, Lei Gao*, Liyun Zhang, Yan Xie, Hanwu Liu, Junping Fan, Ming Wu, Ning Yue, Yan Wang, Meng Han, Tongcan Sun, Qi Ding, Xiyin Zheng, Jidong Cao, Xueqi Shen, Haijun Wang, Tuxunaili Aizitili, Chunyan Wu, Xuehong Wu, Zhenhua Liu, Yiguo Hong, Xiaoguang Lei*, and Yule Liu*
Cell, Available online 8 May 2026
https://doi.org/10.1016/j.cell.2026.04.021
Phytoalexins are core components of plant chemical defense against pathogens. However, the genetic basis and regulatory mechanisms governing their biosynthesis remain preliminary. Debneyol is a well-defined, broad-spectrum fungicidal phytoalexin. Here, we elucidate its biosynthetic pathway, key regulators, and activity against multiple pathogens. We show that debneyol is synthesized from farnesyl pyrophosphate (FPP) through three steps catalyzed by 5-epi-aristolochene synthase (EAS), 5-epi-aristolochene epoxidase (EAE), and epoxide hydrolase-1 (EH1). MCD1 (miR1919-targeted cell death-factor-1) interacts with EAS and EAE, enhancing their association and EAE activity and promoting debneyol biosynthesis. Increased MCD1expression confers plant resistance not only against fungal but also viral and bacterial pathogens. Our work reveals a complete plant phytoalexin-based chemical defense machinery, opening avenues for engineering broad-spectrum plant resistance and industrial-scale debneyol production via synthetic biology.
Development of a clinically viable MRGPRX4 inverse agonist for cholestatic itch treatment
Jun Yang#, Ruichao Shen#, Chunyu Wang#, Wenneng Zhu, Han Ke, Junping Fan, Mengna Zhang, Yingjun Liu, Shuai Li, Guochuan Li, Xiaoming Wang, Yulong Li, Can Cao*, Xiaoguang Lei*
Nature Chemical Biology (2026)
https://doi.org/10.1038/s41589-026-02195-0
Chronic itch, particularly in cholestatic and uremic conditions, poses a notable clinical burden, yet treatment options remain inadequate. MRGPRX4 (hX4), a bile-acid-sensing G-protein-coupled receptor predominantly expressed in human sensory neurons, has emerged as a critical mediator of cholestatic pruritus. Here we identified and characterized HEP-50768, a potent and selective small-molecule inverse agonist of hX4 through high-throughput screening and structure–activity optimization. Structural elucidation through cryo-electron microscopy of the hX4–inverse agonist complex structure revealed the unique binding mode and inhibitory mechanism of HEP-50768. In hX4-humanized rats, HEP-50768 robustly suppressed bile-acid-induced pruritic behaviors. Comprehensive preclinical absorption, distribution, metabolism, excretion and safety profiling was performed in both rats and monkeys, and these findings establish HEP-50768 as a promising therapeutic candidate for chronic itch, supporting its advancement to clinical evaluation.
Structural basis of glucosinolate recognition and transport by plant GTR1
Rui Yan#, Junping Fan#, Cheng Chi#, Bowen Zhang, Di Wu, Huiwen Chen, Jianke Gong*, Xiaoguang Lei*and Daohua Jiang*
Cell Discovery 12, 26 (2026);
https://doi.org/10.1038/s41421-026-00884-7
Glucosinolates (GLSs) play crucial roles in plant defense against herbivores. GTR1 facilitates the high-affinity transport of GLSs through a proton-dependent process. However, the molecular mechanism underlying GLS recognition and transport by GTR1 remains largely unknown. Here, we present four cryo-EM structures of Arabidopsis GTR1 in distinct states, namely, the outward-apo, inward-apo, 4MTB-bound and 3IMG-bound forms, revealing the structural basis for GLS and proton cotransport by GTR1. GTR1 consists of an MFS-like transmembrane domain and an intracellular domain (ICD). The ICD plays an essential role in GTR1 function by interacting with the gating helix, transmembrane helix 7. GLSs are recognized by the central cavity residues and directly interact with the conserved E1X1X2E2K motif. Our structural and functional analyses demonstrated that the E1X1X2E2K motif and Glu513 determine the proton coupling of GTR1. This study provides mechanistic insights into how GTR1 transports GLSs, which could aid in improving crop quality and enhancing resistance to herbivory.
Engineered aldehyde dehydrogenases for amide bond formation
Lei Gao#, Xiang Qiu#, Jun Yang#, Kangdelong Hu#, Peilin Li, Wei Li, Feng Gao, Fabrice Gallou, Florian Kleinbeck, Xiaoguang Lei*
Science 391,eadw3365 (2026)
https://doi.org/10.1126/science.adw336
Amide bond formation is widely used in pharmaceutical synthesis, typically involving stoichiometric coupling reagents to activate carboxylic acid substrates for a condensation reaction. As an alternative approach, we repurposed aldehyde dehydrogenases into oxidative amidases by creating a more hydrophobic and spacious catalytic pocket for amines to capture the thioester intermediate. This biocatalyst efficiently facilitates the formation of amide bonds between diverse aldehydes and amines. We also developed a two-step enzymatic cascade to synthesize amides from broadly available aliphatic alcohols. This biocatalytic strategy enabled the redesign of synthetic routes for five drug molecules. Our findings highlight the potential of oxidative amidases in advancing the synthesis of structurally diverse drug molecules through efficient amide bond formation.
Structure and mechanism of the human bile acid transporter OSTα–OSTβ
Ke Wang, Junping Fan, Huiwen Chen, Bo Huang, Cheng Chi, Rui Yan, Di Wu, Feng Zhou, Wenhua Zhang, Juquan Jiang, Xiaoguang Lei & Daohua Jiang
Nature 651, 251–259 (2026)
https://doi.org/10.1038/s41586-025-09934-8
Bile acids (BAs) are crucial amphipathic surfactants that function as multifaceted regulators in various physiological processes, including nutrient absorption and distribution, lipid metabolism and inflammation. The human organic solute transporter αβ (OSTα–OSTβ; hereafter referred to as OSTα/β) is a BA transporter that has a key role in the secretion and distribution of BAs. Pathogenic mutations in OSTα/β have been associated with cholestasis. Despite the functional importance of OSTα/β in BA homeostasis, the stoichiometry and assembly of the complex and the molecular mechanism that underlies BA transport by OSTα/β remain unknown. Here we present cryo-electron microscopy structures of human OSTα/β in complex with cholesterols and an endogenous substrate, elucidating the structural basis for the function of OSTα/β. OSTα/β is assembled in a novel dimer-of-heterodimers manner: two OSTα units form the homodimeric core, with two OSTβ units bound to the periphery. OSTα adopts the G-protein-coupled-receptor (GPCR) fold and contains a unique cysteine-rich loop with seven palmitoylation sites; these cooperate with transmembrane helices 5 and 6, constituting a BA recognition site. A positive cavity in OSTα connects the BA site and facilitates the transmembrane translocation of BAs through OSTα/β. Together, this study reveals the architecture and transport mechanism of OSTα/β and provides insights into the structure–function relationships of this crucial transporter in BA homeostasis.
CDK10 suppresses nucleic acid sensors-mediated antitumor immunity
Gaoshan Xu, Fusheng Guo, Chuan He, Xiyong Wang, Bolin Xiang, Lifang Fan, Baoxiang Chen, Jiakun Peng, Yishuang Sun, Jie Shi, Xixin Xing, Yingmeng Yao, Panpan Dai, Haiou Li, Wenjun Xiong, Hudan Liu, Rui Xiao, Guoliang Qing, Congqing Jiang, Baishan Jiang, Xiaoguang Lei* & Jinfang Zhang*
Nature Cancer 7, 283–303 (2026)
https://doi.org/10.1038/s43018-025-01100-3
Cancer immunotherapies have revolutionized cancer treatment, yet many patients fail to respond. Activating innate immunity offers a promising approach to enhance therapeutic efficacy, but the signaling kinases directly regulating this process to boost antitumor responses remain elusive. Here we conduct an in vivo kinome CRISPR screen and identify CDK10 as a key suppressor of tumor immune surveillance. Mechanistically, CDK10 phosphorylates DNMT1 and RAP80 to reduce the accumulation of double-stranded RNA and R-loops, which alleviates the activation of innate immune pathways mediated by MDA5 and cGAS. Kinase inhibitor screens identify NVP-AST487 and ponatinib as selective CDK10 inhibitors. Both genetic and pharmacological inhibition of CDK10 activates MDA5 and cGAS pathways, fostering an immunoactive tumor microenvironment that enhances cancer immunotherapy in multiple mouse tumor models. Clinically, low CDK10 expression in tumors correlates with better immunotherapy responses. These findings establish CDK10 as a pivotal modulator of tumor immunity and a potential therapeutic target.
Unveiling FERONIA receptor kinase–mediated cellular mechanisms with a small-molecule inhibitor
Mengze Sun , Baiyan Lu, Ying Yang, Junping Fan, Weiwei Ren, Xiaonan Chu, Yihui Gao, Jun Wu, Jue Wang, Han Ke, Zhiwen Liu, Shaojun Dai, Xiaoguang Lei*, and Chao Li*
PNAS 122(45), e2515322122 (2025)
https://doi.org/10.1073/pnas.2515322122
Since its initial identification as the receptor for Rapid Alkalinization Factor 1 (RALF1), FERONIA (FER) receptor kinase has emerged as a central signaling hub coordi nating plant development, stress adaptation, and immune responses. Nevertheless, fundamental questions persist regarding the precise mechanisms of FER-mediated signal transduction and its context-dependent functional specialization in multicel lular processes. Here, we develop Ferovicin (FRV), a small-molecule inhibitor that specifically disrupts FER kinase activity, thereby enabling mechanistic dissection of FER. Cocrystallization and mutational analysis show that FRV selectively binds to the ATP-binding pocket of the kinase domain of FER and inhibits its kinase activity. Assisted by the FRV tool and quantitative phosphoproteomics, we characterized a series of signaling pathways and networks regulated by RALF1 and FER. Notably, our analysis reveals that RALF1 activates FER through phosphorylation at Ser695, which subsequently inhibits H+ -ATPase1/2 via phosphorylation at Ser899. This mechanism leads to apoplastic alkalinization and regulates cell expansion in the root meristem. Given the conservation of FRV binding sites in FER proteins across land plant species, FRV will serve as a valuable tool for dissecting FER signaling mechanisms as well as facilitating agricultural applications.
Quantitative Reactivity Profiling of Functional Arginine Residues in Human Cancer Cell Line Proteomes
Wenbo Zhao+, Yuliang Tang+, Yihui Gao, Qi Ding, Qiang Li, Wenyang Li, and Xiaoguang Lei*
Angew. Chem. Int. Ed. 64, e202515603 (2025)
https://doi.org/10.1002/anie.202515603
Arginine, a critical amino acid for protein structure and function, is involved in enzyme catalysis and macromolecular interactions. However, selectively targeting its reactive guanidine group has been challenging. Here, we utilized a probe, AP-1, based on phenylglyoxal, which demonstrated remarkable chemical selectivity and reactivity toward arginine residues. Using activity-based protein profifiling (ABPP), we explored the human proteome across four cancer cell lines, obtaining quantitative data for approximately 17 000 arginine residues. This analysis led to the identifification of several previously unreported hyperreactive arginine residues, including R43 of PKM, R171 of LDHA, R172 of LDHB, R341 of CKB, R168 of EIF4A1, and R118 of FUBP1, which are crucial for protein function. Notably, the mutation of CKB’s R341 inhibited cell proliferation and migration by downregulating energy supply. We also introduced ArGO-LDHA-1, a covalent inhibitor targeting LDHA’s hyperreactive arginine residues, showing potential to
enhance chemotherapy effificacy. This work highlights the biological signifificance of arginine residues and provides a platform for large-scale profifiling of arginine reactivity.
Aspartic acid residues in BBE-like enzymes from Morus alba promote a function shift from oxidative cyclization to dehydrogenation
Nianxin Guo, Jun Gu, Qingyang Zhou, Fang Liu, Haoran Dong, Qi Ding, Qixuan Wang, Dongshan Wu, Jun Yang, Junping Fan, Lei Gao*, Kendall N. Houk*, and Xiaoguang Lei*
PNAS 122 (34), e2504346122 (2025)
https://doi.org/10.1073/pnas.2504346122
Berberine bridge enzyme (BBE)-like enzymes catalyze various oxidative cyclization and dehydrogenation reactions in natural product biosynthesis, but the molecular mechanism underlying the selectivity remains unknown. Here, we elucidated the catalytic mechanism of BBE-like oxidases from Morus alba involved in the oxidative cyclization and dehydrogenation of moracin C. X-ray crystal structures of a functionally promiscuous flavin adenine dinucleotide (FAD)–bound oxidase, MaDS1, with and without an oxidative dehydrogenation product were determined at 2.03 Å and 2.21 Å resolution, respectively. Structure-guided mutagenesis and sequence analysis have identified a conserved aspartic acid that directs the reaction toward the oxidative dehydrogenation pathway. A combination of density functional theory (DFT) calculations and molecular dynamics (MD) simulations has revealed that aspartic acid acts as the catalytic base to deprotonate the carbon-cation intermediate to generate the dehydrogenated product, which otherwise undergoes a spontaneous 6π electrocyclization in the oxidative cyclization pathway to furnish the 2H-benzopyran product.