Xiaoguang Lei, Jin Wang, Jianxiong Zhao, Zhenyang Yu, Siyuan Wang, Fusheng Guo, Jun Yang, and Lei Gao
Angew. Chem. Int. Ed. 2024, e202414340
The bisbenzylisoquinoline alkaloids (bisBIAs) have attracted tremendous attention from the synthetic community due to their diverse and intriguing biological activities. Herein, we report the convergent and modular chemoenzymatic syntheses of eight bisBIAs bearing various substitutes and linkages in 5-7 steps. The gram-scale synthesis of various well-designed enantiopure benzylisoquinoline monomers was accomplished via an enzymatic stereoselective Pictet–Spengler reaction, followed by regioselective enzymatic methylation or chemical functionalizations in a sequential one-pot process. A modified intermolecular copper-mediated Ullmann coupling enabled the concise and efficient total synthesis of five different linear bisBIAs with either head-to-tail or tail-to-tail linkage. A biomimetic oxidative phenol dimerization selectively formed the sterically hindered, electron-rich diaryl ether bond, and subsequent intramolecular Suzuki–Miyaura domino reaction or Ullmann coupling facilitated the first enantioselective total synthesis of three macrocyclic bisBIAs, including ent-isogranjine, tetrandrine and O-methylrepandine. This study highlights the great potential of chemoenzymatic strategies in the total synthesis of diverse bisBIAs and paves the way to further explore the biological functions of these natural products.
Junping Fan, Han Ke, Jing Lei, Jin Wang, Makoto Tominaga & Xiaoguang Lei.
Nature Communications, 2024, 15, 6689.
Transient Receptor Potential Vanilloid 1 (TRPV1) plays a central role in pain sensation and is thus an attractive pharmacological drug target. SAF312 is a potent, selective, and non-competitive antagonist of TRPV1 and shows promising potential in treating ocular surface pain. However, the precise mechanism by which SAF312 inhibits TRPV1 remains poorly understood. Here, we present the cryo-EM structure of human TRPV1 in complex with SAF312, elucidating the structural foundation of its antagonistic effects on TRPV1. SAF312 binds to the vanilloid binding pocket, preventing conformational changes in S4 and S5 helices, which are essential for channel gating. Unexpectedly, a putative cholesterol was found to contribute to SAF312’s inhibition. Complemented by mutagenesis experiments and molecular dynamics simulations, our research offers substantial mechanistic insights into the regulation of TRPV1 by SAF312, highlighting the interplay between the antagonist and cholesterol in modulating TRPV1 function. This work not only expands our understanding of TRPV1 inhibition by SAF312 but also lays the groundwork for further developments in the design and optimization of TRPV1-related therapies.
The first total synthesis of (+)-epicolidine C has been accomplished via a late-stage HfCl4-mediated epoxide opening from (+)-PF1052. The 6/6/6/5 tetracyclic core of spylidone has also been constructed from (+)-AB4015-B via late-stage iodine(I)- or manganese(III)-mediated oxidative cyclization reactions, whose absolute stereostructure was unambiguously confirmed by X-ray crystallographic analysis.
Lei Gao, Qi Ding, Xiaoguang Lei* Accounts of Chemical Research, doi：10.1021/acs.accounts.4c00315
The Diels–Alder reaction is well known as a concerted [4 + 2] cycloaddition governed by the Woodward–Hoffmann rules. Since Prof. Otto Diels and his student Kurt Alder initially reported the intermolecular [4 + 2] cycloaddition between cyclopentadiene and quinone in 1928, it has been recognized as one of the most powerful chemical transformations to build C–C bonds and construct cyclic structures. This named reaction has been widely used in synthesizing natural products and drug molecules. Driven by the synthetic importance of the Diels–Alder reaction, identifying the enzyme that stereoselectively catalyzes the Diels–Alder reaction has become an intriguing research area in natural product biosynthesis and biocatalysis. With significant progress in sequencing and bioinformatics, dozens of Diels–Alderases have been characterized in microbial natural product biosynthesis. However, few are evolutionally dedicated to catalyzing an intermolecular Diels–Alder reaction with a concerted mechanism.
The nonselective calcium-permeable Transient Receptor Potential Cation Channel Subfamily V Member4 (TRPV4) channel regulates various physiological activities. Dysfunction of TRPV4 is linked to many severe diseases, including edema, pain, gastrointestinal disorders, lung diseases, and inherited neurodegeneration. Emerging TRPV4 antagonists show potential clinical benefits. However, the molecular mechanisms of TRPV4 antagonism remain poorly understood. Here, cryo-electron microscopy (cryo-EM) structures of human TRPV4 are presented in-complex with two potent antagonists, revealing the detailed binding pockets and regulatory mechanisms of TRPV4 gating. Both antagonists bind to the voltage-sensing-like domain (VSLD) and stabilize the channel in closed states. These two antagonists induce TRPV4 to undergo an apparent fourfold to twofold symmetry transition. Moreover, it is demonstrated that one of the antagonists binds to the VSLD extended pocket, which differs from the canonical VSLD pocket. Complemented with functional and molecular dynamics simulation results, this study provides crucial mechanistic insights into TRPV4 regulation by small-molecule antagonists, which may facilitate future drug discovery targeting TRPV4.
Haoran Dong, Nianxin Guo , Dachao Hu, Benke Hong, Daohong Liao, Hong Jie Zhu, Zhang Yuan Yan, Hui Ming Ge & Xiaoguang Lei
Nature Synthesis. Published online: 25 June 2024. doi: 10.1038/s44160-024-00577-7
Alchivemycin A belongs to a unique class of polyketide natural products isolated from plant-derived actinomycete Streptomyces. It shows potent antibacterial activity and anti-tumour activity. However, its inherent structural complexity and high oxidation state, especially the 2H-tetrahydro-4,6-dioxo-1,2-oxazine (TDO) ring system, present synthetic challenges. Here we report the total synthesis of alchivemycin A using a chemoenzymatic approach that combines de novo skeleton construction and late-stage enzymatic oxidation reactions. The convergent synthesis of the highly functionalized unnatural tetramic acid-bearing intermediate is achieved by boron-alkyl Suzuki−Miyaura cross-coupling, macrolactamization and Lacey–Dieckmann condensation reactions. Efficient enzymatic epoxidations using the redox enzymes AvmO3 and AvmO2 allow rapid access to the desired diepoxide product regio- and stereoselectively. Subsequently, a flavin adenine dinucleotide-dependent enzyme AvmO1 variant optimized via rational protein engineering, AvmO1-Y282R, was used to convert the tetramic acid ring into the TDO ring through a Baeyer–Villiger-type transformation, completing the chemoenzymatic synthesis of alchivemycin A. This work paves the way to further explore the biological functions of alchivemycin A and highlights the utility of chemoenzymatic strategies to tackle synthetic challenges in complex molecule synthesis.
Bioorganic & Medicinal Chemistry，Volume 110, August 2024, 117793
The pathogenic role of anti-phospholipase A2 receptor (PLA2R) antibodies in primary membranous nephropathy (MN) has been well-established. This study aimed to identify potential small-molecule inhibitors against the PLA2R-antibody interaction, offering potential therapeutic benefits. A comprehensive screening of over 4000 small-molecule compounds was conducted by ELISA to assess their inhibitory effects on the binding between the immobilized full-length extracellular PLA2R and its antibodies. The affinity of anti-PLA2R IgG from MN patients and the inhibitory efficacy of each compound were evaluated via surface plasmon resonance (SPR). Human podocyte injuries were analyzed using CCK-8 assay, wound healing assay, western blot analysis, and immunofluorescence, after exposure to MN plasma +/- blocking compound. Fifteen compounds were identified as potential inhibitors, demonstrating inhibition rates >20 % for the PLA2R-antibody interaction. Anti-PLA2R IgG exhibited a consistent affinity among patients (KD = 10−8 M). Macrocarpal B emerged as the most potent inhibitor, reducing the antigen–antibody interaction by nearly 30 % in a dose-dependent manner, comparable to the performance of the 31-mer peptide from the CysR domain. Macrocarpal B bound to the immobilized PLA2R with an affinity of 1.47 × 10−6 M, while showing no binding to anti-PLA2R IgG. Human podocytes exposed to MN plasma showed decreased podocin expression, impaired migration function, and reduced cell viability. Macrocarpal B inhibited the binding of anti-PLA2R IgG to podocytes and reduced the cellular injuries.
New adjuvants that trigger cellular immune responses are urgently needed for the effective development of cancer and virus vaccines. Motivated by recent discoveries that show activation of type I interferon (IFN-I) signaling boosts T cell immunity, this study proposes that targeting this pathway can be a strategic approach to identify novel vaccine adjuvants. Consequently, a comprehensive chemical screening of 6,800 small molecules is performed, which results in the discovery of the natural compound picrasidine S (PS) as an IFN-I inducer. Further analysis reveals that PS acts as a powerful adjuvant, significantly enhancing both humoral and cellular immune responses. At the molecular level, PS initiates the activation of the cGAS-IFN-I pathway, leading to an enhanced T cell response. PS vaccination notably increases the population of CD8+ central memory (TCM)-like cells and boosts the CD8+ T cell-mediated anti-tumor immune response. Thus, this study identifies PS as a promising candidate for developing vaccine adjuvants in cancer prevention.
Haowen Zhang, Shuhan Xie, Jun Yang, Ning Ye, Feng Gao, Fabrice Gallou, Lei Gao, Xiaoguang Lei
Angewandte Chemie International Edition Pub Date : 2024-05-15 , DOI: 10.1002/anie.202405833
Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.
Qi Ding, Nianxin Guo, Lei Gao, Michelle McKee, Dongshan Wu, Jun Yang, Junping Fan, Jing-Ke Weng & Xiaoguang Lei Nature Communications ，volume 15, Article number: 2492 (2024) Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.
-
-
-
-
-
-
-
-
-
-
Concise and Modular Chemoenzymatic Total Synthesis of Bisbenzylisoquinoline Alkaloids
Xiaoguang Lei, Jin Wang, Jianxiong Zhao, Zhenyang Yu,
Siyuan Wang, Fusheng Guo, Jun Yang, and Lei Gao
Angew. Chem. Int. Ed. 2024, e202414340
The bisbenzylisoquinoline alkaloids (bisBIAs) have attracted tremendous attention from the synthetic community due to their diverse and intriguing biological activities. Herein, we report the convergent and modular chemoenzymatic syntheses of eight bisBIAs bearing various substitutes and linkages in 5-7 steps. The gram-scale synthesis of various well-designed enantiopure benzylisoquinoline monomers was accomplished via an enzymatic stereoselective Pictet–Spengler reaction, followed by regioselective enzymatic methylation or chemical functionalizations in a sequential one-pot process. A modified intermolecular copper-mediated Ullmann coupling enabled the concise and efficient total synthesis of five different linear bisBIAs with either head-to-tail or tail-to-tail linkage. A biomimetic oxidative phenol dimerization selectively formed the sterically hindered, electron-rich diaryl ether bond, and subsequent intramolecular Suzuki–Miyaura domino reaction or Ullmann coupling facilitated the first enantioselective total synthesis of three macrocyclic bisBIAs, including ent-isogranjine, tetrandrine and O-methylrepandine. This study highlights the great potential of chemoenzymatic strategies in the total synthesis of diverse bisBIAs and paves the way to further explore the biological functions of these natural products.
Structural basis of TRPV1 inhibition by SAF312 and cholesterol
Junping Fan, Han Ke, Jing Lei, Jin Wang, Makoto Tominaga & Xiaoguang Lei.
Nature Communications, 2024, 15, 6689.
Transient Receptor Potential Vanilloid 1 (TRPV1) plays a central role in pain sensation and is thus an attractive pharmacological drug target. SAF312 is a potent, selective, and non-competitive antagonist of TRPV1 and shows promising potential in treating ocular surface pain. However, the precise mechanism by which SAF312 inhibits TRPV1 remains poorly understood. Here, we present the cryo-EM structure of human TRPV1 in complex with SAF312, elucidating the structural foundation of its antagonistic effects on TRPV1. SAF312 binds to the vanilloid binding pocket, preventing conformational changes in S4 and S5 helices, which are essential for channel gating. Unexpectedly, a putative cholesterol was found to contribute to SAF312’s inhibition. Complemented by mutagenesis experiments and molecular dynamics simulations, our research offers substantial mechanistic insights into the regulation of TRPV1 by SAF312, highlighting the interplay between the antagonist and cholesterol in modulating TRPV1 function. This work not only expands our understanding of TRPV1 inhibition by SAF312 but also lays the groundwork for further developments in the design and optimization of TRPV1-related therapies.
Synthesis of (+)-epicolidine C and the 6/6/6/5 tetracyclic core of spylidone
Haoran Dong, Xiaoguang Lei
Tetrahedron Letters, 2024, 146, 155181.
The first total synthesis of (+)-epicolidine C has been accomplished via a late-stage HfCl4-mediated epoxide opening from (+)-PF1052. The 6/6/6/5 tetracyclic core of spylidone has also been constructed from (+)-AB4015-B via late-stage iodine(I)- or manganese(III)-mediated oxidative cyclization reactions, whose absolute stereostructure was unambiguously confirmed by X-ray crystallographic analysis.
Hunting for the Intermolecular Diels−Alderase
Lei Gao, Qi Ding, Xiaoguang Lei*
Accounts of Chemical Research, doi：10.1021/acs.accounts.4c00315
The Diels–Alder reaction is well known as a concerted [4 + 2] cycloaddition governed by the Woodward–Hoffmann rules. Since Prof. Otto Diels and his student Kurt Alder initially reported the intermolecular [4 + 2] cycloaddition between cyclopentadiene and quinone in 1928, it has been recognized as one of the most powerful chemical transformations to build C–C bonds and construct cyclic structures. This named reaction has been widely used in synthesizing natural products and drug molecules. Driven by the synthetic importance of the Diels–Alder reaction, identifying the enzyme that stereoselectively catalyzes the Diels–Alder reaction has become an intriguing research area in natural product biosynthesis and biocatalysis. With significant progress in sequencing and bioinformatics, dozens of Diels–Alderases have been characterized in microbial natural product biosynthesis. However, few are evolutionally dedicated to catalyzing an intermolecular Diels–Alder reaction with a concerted mechanism.
Structural Pharmacology of TRPV4 Antagonists
Junping Fan, Chang Guo, Daohong Liao, Han Ke, Jing Lei, Wenjun Xie, Yuliang Tang, Makoto Tominaga, Zhuo Huang, Xiaoguang Lei
Advanced Science 2024, e2401583. doi: 10.1002/advs.202401583
The nonselective calcium-permeable Transient Receptor Potential Cation Channel Subfamily V Member4 (TRPV4) channel regulates various physiological activities. Dysfunction of TRPV4 is linked to many severe diseases, including edema, pain, gastrointestinal disorders, lung diseases, and inherited neurodegeneration. Emerging TRPV4 antagonists show potential clinical benefits. However, the molecular mechanisms of TRPV4 antagonism remain poorly understood. Here, cryo-electron microscopy (cryo-EM) structures of human TRPV4 are presented in-complex with two potent antagonists, revealing the detailed binding pockets and regulatory mechanisms of TRPV4 gating. Both antagonists bind to the voltage-sensing-like domain (VSLD) and stabilize the channel in closed states. These two antagonists induce TRPV4 to undergo an apparent fourfold to twofold symmetry transition. Moreover, it is demonstrated that one of the antagonists binds to the VSLD extended pocket, which differs from the canonical VSLD pocket. Complemented with functional and molecular dynamics simulation results, this study provides crucial mechanistic insights into TRPV4 regulation by small-molecule antagonists, which may facilitate future drug discovery targeting TRPV4.
Chemoenzymatic total synthesis of alchivemycin A
Haoran Dong, Nianxin Guo , Dachao Hu, Benke Hong, Daohong Liao, Hong Jie Zhu, Zhang Yuan Yan, Hui Ming Ge & Xiaoguang Lei
Nature Synthesis. Published online: 25 June 2024.
doi: 10.1038/s44160-024-00577-7
Alchivemycin A belongs to a unique class of polyketide natural products isolated from plant-derived actinomycete Streptomyces. It shows potent antibacterial activity and anti-tumour activity. However, its inherent structural complexity and high oxidation state, especially the 2H-tetrahydro-4,6-dioxo-1,2-oxazine (TDO) ring system, present synthetic challenges. Here we report the total synthesis of alchivemycin A using a chemoenzymatic approach that combines de novo skeleton construction and late-stage enzymatic oxidation reactions. The convergent synthesis of the highly functionalized unnatural tetramic acid-bearing intermediate is achieved by boron-alkyl Suzuki−Miyaura cross-coupling, macrolactamization and Lacey–Dieckmann condensation reactions. Efficient enzymatic epoxidations using the redox enzymes AvmO3 and AvmO2 allow rapid access to the desired diepoxide product regio- and stereoselectively. Subsequently, a flavin adenine dinucleotide-dependent enzyme AvmO1 variant optimized via rational protein engineering, AvmO1-Y282R, was used to convert the tetramic acid ring into the TDO ring through a Baeyer–Villiger-type transformation, completing the chemoenzymatic synthesis of alchivemycin A. This work paves the way to further explore the biological functions of alchivemycin A and highlights the utility of chemoenzymatic strategies to tackle synthetic challenges in complex molecule synthesis.
Macrocarpal B blocks the binding between the phospholipase A2 receptor and its antibodies
Zixin Feng, Fu-sheng Guo, Qian Wang, Miao Wang, Ming-Hui Zhao, Zhao Cui, Xiaoguang Lei
Bioorganic & Medicinal Chemistry，Volume 110, August 2024, 117793
The pathogenic role of anti-phospholipase A2 receptor (PLA2R) antibodies in primary membranous nephropathy (MN) has been well-established. This study aimed to identify potential small-molecule inhibitors against the PLA2R-antibody interaction, offering potential therapeutic benefits. A comprehensive screening of over 4000 small-molecule compounds was conducted by ELISA to assess their inhibitory effects on the binding between the immobilized full-length extracellular PLA2R and its antibodies. The affinity of anti-PLA2R IgG from MN patients and the inhibitory efficacy of each compound were evaluated via surface plasmon resonance (SPR). Human podocyte injuries were analyzed using CCK-8 assay, wound healing assay, western blot analysis, and immunofluorescence, after exposure to MN plasma +/- blocking compound. Fifteen compounds were identified as potential inhibitors, demonstrating inhibition rates >20 % for the PLA2R-antibody interaction. Anti-PLA2R IgG exhibited a consistent affinity among patients (KD = 10−8 M). Macrocarpal B emerged as the most potent inhibitor, reducing the antigen–antibody interaction by nearly 30 % in a dose-dependent manner, comparable to the performance of the 31-mer peptide from the CysR domain. Macrocarpal B bound to the immobilized PLA2R with an affinity of 1.47 × 10−6 M, while showing no binding to anti-PLA2R IgG. Human podocytes exposed to MN plasma showed decreased podocin expression, impaired migration function, and reduced cell viability. Macrocarpal B inhibited the binding of anti-PLA2R IgG to podocytes and reduced the cellular injuries.
Picrasidine S Induces cGAS-Mediated Cellular Immune Response as a Novel Vaccine Adjuvant
Xiaofan Ding, Mengxue Sun, Fusheng Guo, Xinmin Qian, Haoyu Yuan, Wenjiao Lou, Qixuan Wang, Xiaoguang Lei, Wenwen Zeng
Advanced science (Weinheim, Baden-Wurttemberg, Germany), e2310108. Advance online publication. https://doi.org/10.1002/advs.202310108
New adjuvants that trigger cellular immune responses are urgently needed for the effective development of cancer and virus vaccines. Motivated by recent discoveries that show activation of type I interferon (IFN-I) signaling boosts T cell immunity, this study proposes that targeting this pathway can be a strategic approach to identify novel vaccine adjuvants. Consequently, a comprehensive chemical screening of 6,800 small molecules is performed, which results in the discovery of the natural compound picrasidine S (PS) as an IFN-I inducer. Further analysis reveals that PS acts as a powerful adjuvant, significantly enhancing both humoral and cellular immune responses. At the molecular level, PS initiates the activation of the cGAS-IFN-I pathway, leading to an enhanced T cell response. PS vaccination notably increases the population of CD8+ central memory (TCM)-like cells and boosts the CD8+ T cell-mediated anti-tumor immune response. Thus, this study identifies PS as a promising candidate for developing vaccine adjuvants in cancer prevention.
Chemoenzymatic Synthesis of 2-Aryl Thiazolines from 4- Hydroxybenzaldehydes Using Vanillyl Alcohol Oxidases
Haowen Zhang, Shuhan Xie, Jun Yang, Ning Ye, Feng Gao, Fabrice Gallou, Lei Gao, Xiaoguang Lei
Angewandte Chemie International Edition Pub Date : 2024-05-15 , DOI: 10.1002/anie.202405833
Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis.
The evolutionary origin of naturally occurring intermolecular Diels-Alderases from Morus alba
Qi Ding, Nianxin Guo, Lei Gao, Michelle McKee, Dongshan Wu, Jun Yang, Junping Fan, Jing-Ke Weng & Xiaoguang Lei
Nature Communications ，volume 15, Article number: 2492 (2024)
Biosynthetic enzymes evolutionarily gain novel functions, thereby expanding the structural diversity of natural products to the benefit of host organisms. Diels-Alderases (DAs), functionally unique enzymes catalysing [4 + 2] cycloaddition reactions, have received considerable research interest. However, their evolutionary mechanisms remain obscure. Here, we investigate the evolutionary origins of the intermolecular DAs in the biosynthesis of Moraceae plant-derived Diels-Alder-type secondary metabolites. Our findings suggest that these DAs have evolved from an ancestor functioning as a flavin adenine dinucleotide (FAD)-dependent oxidocyclase (OC), which catalyses the oxidative cyclisation reactions of isoprenoid-substituted phenolic compounds. Through crystal structure determination, computational calculations, and site-directed mutagenesis experiments, we identified several critical substitutions, including S348L, A357L, D389E and H418R that alter the substrate-binding mode and enable the OCs to gain intermolecular DA activity during evolution. This work provides mechanistic insights into the evolutionary rationale of DAs and paves the way for mining and engineering new DAs from other protein families.