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Wen Liu - One of the best experts on this subject based on the ideXlab platform.
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mutational biosynthesis to generate novel analogs of Nosiheptide featuring a fluorinated indolic acid moiety
Organic and Biomolecular Chemistry, 2020Co-Authors: E Zhang, Shoufeng Wang, Heng Guo, Yafei Fan, Wengui Wang, Dandan Chen, Qian Yang, Yu Yin, Daijie Chen, Wen LiuAbstract:Nosiheptide (NOS) is a member of bicyclic thiopeptides possessing a biologically important indolic acid (IA) moiety appended onto the family-characteristic core system. The IA formation relies primarily on NosL, a radical S-adenosylmethionine (SAM) protein that catalyzes a complex rearrangement of the carbon side chain of l-tryptophan, leading to the generation of 3-methyl-2-indolic acid (MIA). Here, we establish an efficient mutational biosynthesis strategy for the structural expansion of the side-ring system of NOS. The nosL-deficient mutant Streptomyces actuosus SL4005 complemented by chemically feeding 6-fluoro-MIA is capable of accumulating two new products. The target product 6'-fluoro-NOS contains an additional fluorine atom at C6 of the IA moiety, in contrast with an unexpected product 6'-fluoro-NOSint that features an open side ring and a bis-dehydroalanine (Dha) tail. The newly obtained 6'-fluoro-NOS displayed equivalent or slightly reduced activities against the tested drug-resistant pathogens compared with NOS, but dramatically decreased water solubility compared with NOS. Our results indicate that the modification of the IA moiety of NOS not only affects its biological activity but also affects its activity which will be key considerations for further modification.
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isolation and structure determination of two new Nosiheptide type compounds provide insights into the function of the cytochrome p450 oxygenase nocv in nocathiacin biosynthesis
Organic chemistry frontiers, 2020Co-Authors: Xuebing Bai, Heng Guo, Dandan Chen, Qian Yang, Jiang Tao, Wen LiuAbstract:Thiopeptides, which are a class of sulfur-rich, ribosomally synthesized and post-translationally modified peptides (RiPPs), have great potential in the treatment of diseases caused by oral pathogens. Nocathiacin I (NOC-I) and Nosiheptide (NOS) are two structurally similar thiopeptide members that feature an indolic side ring. In the structure of NOC-I, this side ring is further rigidified through the formation of an ether linkage; however, the related biosynthetic process remains poorly understood. Here, we report that NocV, a cytochrome P450 protein found to be unique in the biosynthetic pathway of NOC-I, is responsible for the establishment of the intramolecular ether linkage through two oxidation steps. This observation benefited from the heterologous overexpression of the gene nocV in an engineered Streptomyces strain producing the bicyclic NOS intermediate NOS1260, and subsequent isolation and structure characterization of two functionalized products. The product NOS-V1 contains a new hydroxyl group at Cα of the residue Glu6, in contrast with the other product NOS-V2, in which this hydroxyl group is further coupled with the C4 methyl group of the indolic moiety to form an ether linkage. These findings provide insights into the catalytic logic of NocV in the biosynthesis of NOC-I, during which this cytochrome P450 protein appears to act in tandem on two positions in NOS1260 by selectively hydroxylating Glu6 and then oxidatively coupling the indolic moiety. Rigidifying the side ring via ether bond formation has a positive impact on the antibacterial properties of NOS-type thiopeptides, evidenced by the improved activity of NOC-V2 against various tested oral pathogens compared with NOS1260.
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radical s adenosylmethionine protein nosn forms the side ring system of Nosiheptide by functionalizing the polythiazolyl peptide s conjugated indolic moiety
Organic Letters, 2019Co-Authors: Yanping Qiu, Shoufeng Wang, Shuaixiang Zhou, Yinlong Guo, Wen LiuAbstract:NosN is a radical S-adenosylmethionine protein observed in the biosynthesis of the bicyclic thiopeptide Nosiheptide. Insights are provided in terms of the timing of NosN action, its catalytic mechanism, and its role in side ring formation. Beyond being a methyltransferase, NosN transforms a polythiazolyl peptide intermediate by functionalizing the S-conjugated indolic moiety to selectively build a C1 unit, form an ester linkage to the thiopeptide framework, and establish the side ring system specific for Nosiheptide.
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Radical S‑Adenosylmethionine Protein NosN Forms the Side Ring System of Nosiheptide by Functionalizing the Polythiazolyl Peptide S‑Conjugated Indolic Moiety
2019Co-Authors: Yanping Qiu, Shoufeng Wang, Shuaixiang Zhou, Yinlong Guo, Wen LiuAbstract:NosN is a radical S-adenosylmethionine protein observed in the biosynthesis of the bicyclic thiopeptide Nosiheptide. Insights are provided in terms of the timing of NosN action, its catalytic mechanism, and its role in side ring formation. Beyond being a methyltransferase, NosN transforms a polythiazolyl peptide intermediate by functionalizing the S-conjugated indolic moiety to selectively build a C1 unit, form an ester linkage to the thiopeptide framework, and establish the side ring system specific for Nosiheptide
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thiolation protein based transfer of indolyl to a ribosomally synthesized polythiazolyl peptide intermediate during the biosynthesis of the side ring system of Nosiheptide
Journal of the American Chemical Society, 2017Co-Authors: Yanping Qiu, Shuaixiang Zhou, Yinlong Guo, Rijing Liao, Fang Zhang, Chao Peng, Wen LiuAbstract:Nosiheptide, a potent bicyclic member of the family of thiopeptide antibiotics, possesses a distinctive l-Trp-derived indolyl moiety. The way in which this moiety is incorporated into a ribosomally synthesized and post-translationally modified thiopeptide remains poorly understood. Here, we report that NosK, an α/β-hydrolase fold protein, mediates the transfer of indolyl from NosJ, a discrete thiolation protein, to a linear pentathiazolyl peptide intermediate rather than its genetically encoded untreated precursor. This intermediate results from enzymatic processing of the peptide precursor, in which five of the six l-Cys residues are transformed into thiazoles but Cys4 selectively remains unmodified for indolyl substitution via a thioester exchange. Determining the timing of indolyl incorporation, which expands the chemical space of a thiopeptide framework, facilitates mechanistic access to the unusual logic of post-translational modifications in the biosynthesis of Nosiheptide-type thiopeptide members t...
Fuli Wang - One of the best experts on this subject based on the ideXlab platform.
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modeling and parameter updating for Nosiheptide fed batch fermentation process
Industrial & Engineering Chemistry Research, 2016Co-Authors: Dapeng Niu, Long Zhang, Fuli WangAbstract:Nosiheptide is a sulfur-containing peptide antibiotic obtained through fermentation. It can be used as feed additives because of its relative safety and good effect. However, Nosiheptide fermentation does not have a high yield. Keeping the fermentation environment or operating conditions optimum through optimization is an effective way to improve Nosiheptide’s yield, while accurate and reliable process models are the basis to achieve process optimization. Based on the reaction mechanism of the Nosiheptide fed-batch fermentation process, we establish its mechanism models. Fermentation processes have slow time-varying characteristics and the conditions usually change due to disturbances during the production process, so the accuracy of established models tends to decline. Thus, models do not match the actual process gradually, leading to model mismatch, which have bad effects on the optimization and control of the process. Therefore, it is necessary to update the process models in time. In this paper, we up...
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optimization of Nosiheptide fed batch fermentation process based on hybrid model
Industrial & Engineering Chemistry Research, 2013Co-Authors: Dapeng Niu, Mingxing Jia, Fuli WangAbstract:Nosiheptide, a sulfur-containing peptide antibiotic obtained through fermentation, is a perfect feed additive, but its yield in industry is not high. Process optimization is a good way to increase Nosiheptide yield, maintaining the optimum operating conditions of the fermentation process, while optimization of the process requires a sufficiently accurate and robust process model. In this paper, the mechanism model for Nosiheptide fed-batch fermentation is first established. Then, in order to improve performance of the mechanism model, a hybrid model is built using least-squares support vector machines to compensate the errors between the mechanism model and the process. The hybrid model not only overcomes pure black-box model’s shortcoming that it often has poor generalization ability but improves the mechanism model’s accuracy. A yield optimization model of Nosiheptide fed-batch fermentation process is then established based on the hybrid model. An improved particle swarm optimization algorithm is used t...
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neural network ensemble modeling for Nosiheptide fermentation process based on partial least squares regression
Chemometrics and Intelligent Laboratory Systems, 2011Co-Authors: Dapeng Niu, Fuli Wang, Lingling Zhang, Mingxing JiaAbstract:Abstract Nosiheptide fermentation product concentration model based on neural network ensemble is presented. Data for building the model is re-sampled from the original training data using Bagging approach. For each pair of training data an individual Elman neural network is trained. Then outputs of the individual neural network are then combined to form the overall output of the neural network ensemble through the weighted average method and the combining weights are determined by partial least squares regression. The model built on neural network ensemble is compared to a single neural network model, and the results show that it has high accuracy and generalization ability.
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the application of adaptive critic design in the Nosiheptide fermentation
International Symposium on Neural Networks, 2007Co-Authors: Dapeng Zhang, Fuli Wang, Zhiling LinAbstract:An adaptive critic design is used in the Nosiheptide fermentation process to solve the intractable optimization problem. The utility function is defined as the increment of biomass concentration at the adjacent intervals. The state variables are chosen as the biomass concentration, the substrate concentration, the dissolved oxygen concentration and the inhibition concentration. The decision variables are chosen as the temperature, the stirring speed, the airflow and the tank pressure. The adaptive critic method determines optimal control laws for a system by successively adapting the critic networks and the action network. The simulation shows at the same initial conditions this technique can make the fermentation shorten 6 hours.
Shoufeng Wang - One of the best experts on this subject based on the ideXlab platform.
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Nosiheptide analogues as potential antibacterial agents via dehydroalanine region modifications semi synthesis antimicrobial activity and molecular docking study
Bioorganic & Medicinal Chemistry, 2021Co-Authors: Yafei Fan, Hangfei Chen, Wengui Wang, Kongkai Zhu, Zhi Ruan, Shoufeng WangAbstract:Abstract The frequent and inappropriate use of antibiotics aggravate the variation and evolution of multidrug-resistant bacteria, posing a serious threat to public health. Nosiheptide (NOS) has excellent lethality against a variety of Gram-positive bacteria, however the physical and chemical drawbacks hamper its routine application in clinical practice. In this study, by using NOS as the starting material, a total of 15 NOS analogues (2a-4e) were semi-synthesized via its dehydroalanine residue reacting with monosubstituted anilines. In vitro antimicrobial susceptibilities of NOS and its analogues against two methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) clinical isolates were determined by broth microdilution assay to determine the minimum inhibitory concentration (MIC). Antimicrobial susceptibility testing data shown that most of the NOS analogues had a better antibacterial effect than the parent compound, with compound 3c exhibiting the highest antibacterial activity against VRE (MIC = 0.0078 mg/L) and MRSA (MIC
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mutational biosynthesis to generate novel analogs of Nosiheptide featuring a fluorinated indolic acid moiety
Organic and Biomolecular Chemistry, 2020Co-Authors: E Zhang, Shoufeng Wang, Heng Guo, Yafei Fan, Wengui Wang, Dandan Chen, Qian Yang, Yu Yin, Daijie Chen, Wen LiuAbstract:Nosiheptide (NOS) is a member of bicyclic thiopeptides possessing a biologically important indolic acid (IA) moiety appended onto the family-characteristic core system. The IA formation relies primarily on NosL, a radical S-adenosylmethionine (SAM) protein that catalyzes a complex rearrangement of the carbon side chain of l-tryptophan, leading to the generation of 3-methyl-2-indolic acid (MIA). Here, we establish an efficient mutational biosynthesis strategy for the structural expansion of the side-ring system of NOS. The nosL-deficient mutant Streptomyces actuosus SL4005 complemented by chemically feeding 6-fluoro-MIA is capable of accumulating two new products. The target product 6'-fluoro-NOS contains an additional fluorine atom at C6 of the IA moiety, in contrast with an unexpected product 6'-fluoro-NOSint that features an open side ring and a bis-dehydroalanine (Dha) tail. The newly obtained 6'-fluoro-NOS displayed equivalent or slightly reduced activities against the tested drug-resistant pathogens compared with NOS, but dramatically decreased water solubility compared with NOS. Our results indicate that the modification of the IA moiety of NOS not only affects its biological activity but also affects its activity which will be key considerations for further modification.
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radical s adenosylmethionine protein nosn forms the side ring system of Nosiheptide by functionalizing the polythiazolyl peptide s conjugated indolic moiety
Organic Letters, 2019Co-Authors: Yanping Qiu, Shoufeng Wang, Shuaixiang Zhou, Yinlong Guo, Wen LiuAbstract:NosN is a radical S-adenosylmethionine protein observed in the biosynthesis of the bicyclic thiopeptide Nosiheptide. Insights are provided in terms of the timing of NosN action, its catalytic mechanism, and its role in side ring formation. Beyond being a methyltransferase, NosN transforms a polythiazolyl peptide intermediate by functionalizing the S-conjugated indolic moiety to selectively build a C1 unit, form an ester linkage to the thiopeptide framework, and establish the side ring system specific for Nosiheptide.
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Radical S‑Adenosylmethionine Protein NosN Forms the Side Ring System of Nosiheptide by Functionalizing the Polythiazolyl Peptide S‑Conjugated Indolic Moiety
2019Co-Authors: Yanping Qiu, Shoufeng Wang, Shuaixiang Zhou, Yinlong Guo, Wen LiuAbstract:NosN is a radical S-adenosylmethionine protein observed in the biosynthesis of the bicyclic thiopeptide Nosiheptide. Insights are provided in terms of the timing of NosN action, its catalytic mechanism, and its role in side ring formation. Beyond being a methyltransferase, NosN transforms a polythiazolyl peptide intermediate by functionalizing the S-conjugated indolic moiety to selectively build a C1 unit, form an ester linkage to the thiopeptide framework, and establish the side ring system specific for Nosiheptide
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opportunities and challenges from current investigations into the biosynthetic logic of Nosiheptide represented thiopeptide antibiotics
Current Opinion in Chemical Biology, 2013Co-Authors: Shoufeng Wang, Shuaixiang Zhou, Wen LiuAbstract:Nosiheptide is an archetypal thiopeptide antibiotic, possessing a characteristic macrocyclic core that contains a 6-membered heterocycle central to multiple azol(in)es and dehydroamino acids. The discovery of the ribosomal origin of thiopeptides revealed a unifying theme, showing that the structural complexity arises from post-translational modifications (PTMs) of precursor peptides. Thiopeptide framework formation proceeds via cyclodehydration/dehydrogenation (for azol(in)es), dehydration (for dehydroamino acids), and cycloaddition (for the central heterocycle domain). This common process has not been reproduced in vitro, partly due to the poorly understood logic of thiopeptide biosynthetic pathways. Utilizing Nosiheptide biosynthesis as a model system, we herein consider how nature coordinates a number of highly interwined, common and specific PTMs to accomplish the complexity of ribosomally synthesized and post-translationally modified peptides.
Squire J Booker - One of the best experts on this subject based on the ideXlab platform.
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capturing intermediates in the reaction catalyzed by nosn a class c radical s adenosylmethionine methylase involved in the biosynthesis of the Nosiheptide side ring system
Journal of the American Chemical Society, 2019Co-Authors: Bo Wang, Joseph W Lamattina, Savannah Marshall, Squire J BookerAbstract:Nosiheptide is a ribosomally synthesized and post-translationally modified thiopeptide natural product that possesses antibacterial, anticancer, and immunosuppressive properties. It contains a bicyclic structure composed of a large macrocycle and a unique side-ring system containing a 3,4-dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the structural peptide, encoded by the nosM gene, the biosynthesis of the side-ring structure requires the actions of NosI, -J, -K, -L, and -N. NosN is annotated as a class C radical S-adenosylmethionine (SAM) methylase, but its true function is to transfer a C1 unit from SAM to C4 of 3-methyl-2-indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. However, exactly when NosN performs its function during the biosynthesis of Nosiheptide is unknown. Herein, we report the syntheses and use of ...
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Capturing Intermediates in the Reaction Catalyzed by NosN, a Class C Radical S‑Adenosylmethionine Methylase Involved in the Biosynthesis of the Nosiheptide Side-Ring System
2019Co-Authors: Bo Wang, Joseph W Lamattina, Savannah L. Marshall, Squire J BookerAbstract:Nosiheptide is a ribosomally synthesized and post-translationally modified thiopeptide natural product that possesses antibacterial, anticancer, and immunosuppressive properties. It contains a bicyclic structure composed of a large macrocycle and a unique side-ring system containing a 3,4-dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the structural peptide, encoded by the nosM gene, the biosynthesis of the side-ring structure requires the actions of NosI, -J, -K, -L, and -N. NosN is annotated as a class C radical S-adenosylmethionine (SAM) methylase, but its true function is to transfer a C1 unit from SAM to C4 of 3-methyl-2-indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. However, exactly when NosN performs its function during the biosynthesis of Nosiheptide is unknown. Herein, we report the syntheses and use of three peptide mimics as potential substrates designed to address the timing of NosN’s function. Our results show that NosN clearly closes the side ring before NosO forms the pyridine ring and most likely before NosD/E catalyzes formation of the dehydrated amino acids, although the possibility of a more random process (i.e., NosN acting after NosD/E) cannot be ruled out. Using a substrate mimic containing a rigid structure, we also identify and characterize two reaction-based adducts containing SAM fused to C4 of MIA. The two SAM adducts are derived from a consensus radical-containing species proposed to be the key intermediateor a derivative of the key intermediatein our proposed catalytic mechanism of NosN
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using peptide mimics to study the biosynthesis of the side ring system of Nosiheptide
Methods in Enzymology, 2018Co-Authors: Bo Wang, Joseph W Lamattina, Edward D Badding, Lauren K Gadsby, Tyler L Grove, Squire J BookerAbstract:Thiopeptide natural products have gained interest recently for their diverse pharmacological properties, including antibacterial, antifungal, anticancer, and antimalarial activities. Due to their inherent poor solubility and uptake, there is interest in developing new thiopeptides that mimic these unique structures, but which exhibit better pharmacokinetic properties. One strategy is to exploit the biosynthetic pathways using a chemoenzymatic approach to make analogs. However, a complete understanding of thiopeptide biosynthesis is not available, especially for those molecules that contain a large number of modifications to the thiopeptide core. This gap in knowledge and the lack of a facile method for generating a variety of thiopeptide intermediates makes studying particular enzymatic steps difficult. We developed a method to produce thiopeptide mimics based on established synthetic procedures to study the reaction catalyzed by NosN, the class C radical S-adenosylmethionine methylase involved in carbon transfer to C4 of 3-methylindolic acid and completion of the side-ring system in Nosiheptide. Herein, we detail strategies for overproducing and isolating NosN, as well as procedures for synthesizing substrate mimics to study the formation of the side-ring system of Nosiheptide.
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nosn a radical s adenosylmethionine methylase catalyzes both c1 transfer and formation of the ester linkage of the side ring system during the biosynthesis of Nosiheptide
Journal of the American Chemical Society, 2017Co-Authors: Joseph W Lamattina, Bo Wang, Edward D Badding, Lauren K Gadsby, Tyler L Grove, Squire J BookerAbstract:Nosiheptide, a member of the e series of macrocyclic thiopeptide natural products, contains a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide backbone via ester and thioester linkages, respectively. Herein, we show that NosN, a predicted class C radical S-adenosylmethionine (SAM) methylase, catalyzes both the transfer of a C1 unit from SAM to 3-methylindolic acid linked to Cys8 of a synthetic substrate surrogate as well as the formation of the ester linkage between Glu6 and the nascent C4 methylene moiety of DMIA. In contrast to previous studies that indicated that 5′-methylthioadenosine is the immediate methyl donor in the reaction, in our studies, SAM itself plays this role, giving rise to S-adenosylhomocysteine as a coproduct of the reaction.
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rerouting the pathway for the biosynthesis of the side ring system of Nosiheptide the roles of nosi nosj and nosk
Journal of the American Chemical Society, 2017Co-Authors: Edward D Badding, Joseph W Lamattina, Lauren K Gadsby, Tyler L Grove, Amie K Boal, Squire J BookerAbstract:Nosiheptide (NOS) is a highly modified thiopeptide antibiotic that displays formidable in vitro activity against a variety of Gram-positive bacteria. In addition to a central hydroxypyridine ring, NOS contains several other modifications, including multiple thiazole rings, dehydro-amino acids, and a 3,4-dimethylindolic acid (DMIA) moiety. The DMIA moiety is required for NOS efficacy and is synthesized from l-tryptophan in a series of reactions that have not been fully elucidated. Herein, we describe the role of NosJ, the product of an unannotated gene in the biosynthetic operon for NOS, as an acyl carrier protein that delivers 3-methylindolic acid (MIA) to NosK. We also reassign the role of NosI as the enzyme responsible for catalyzing the ATP-dependent activation of MIA and MIA’s attachment to the phosphopantetheine moiety of NosJ. Lastly, NosK catalyzes the transfer of the MIA group from NosJ-MIA to a conserved serine residue (Ser102) on NosK. The X-ray crystal structure of NosK, solved to 2.3 A resolut...
Qi Zhang - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of the Nosiheptide indole side ring centers on a cryptic carrier protein nosj
Nature Communications, 2017Co-Authors: Wei Ding, Wanqiu Liu, Junfeng Zhao, Wei Zhang, Zixin Deng, Boping Tang, Qi ZhangAbstract:Nosiheptide is a prototypal thiopeptide antibiotic, containing an indole side ring in addition to its thiopeptide-characteristic macrocylic scaffold. This indole ring is derived from 3-methyl-2-indolic acid (MIA), a product of the radical S-adenosylmethionine enzyme NosL, but how MIA is incorporated into Nosiheptide biosynthesis remains to be investigated. Here we report functional dissection of a series of enzymes involved in Nosiheptide biosynthesis. We show NosI activates MIA and transfers it to the phosphopantetheinyl arm of a carrier protein NosJ. NosN then acts on the NosJ-bound MIA and installs a methyl group on the indole C4, and the resulting dimethylindolyl moiety is released from NosJ by a hydrolase-like enzyme NosK. Surface plasmon resonance analysis show that the molecular complex of NosJ with NosN is much more stable than those with other enzymes, revealing an elegant biosynthetic strategy in which the reaction flux is controlled by protein-protein interactions with different binding affinities.Thiopeptides such as Nosiheptide are clinically-interesting antimicrobial natural products. Here the authors show the functional dissection of a series of enzymes involved in Nosiheptide biosynthesis, revealing a unique biosynthetic pathway that centers on a previously-unknown carrier protein.
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nucleoside linked shunt products in the reaction catalyzed by the class c radical s adenosylmethionine methyltransferase nosn
Chemical Communications, 2017Co-Authors: Wei Ding, Zixin Deng, Haocheng Qianzhu, Fener Chen, Qi ZhangAbstract:NosN is a class C radical S-adenosylmethionine (SAM) methyltransferase (RSMT) involved in the biosynthesis of Nosiheptide, a clinically interesting thiopeptide antibiotic produced by Streptomyces actuosus. NosN employs an unprecedented catalytic mechanism, in which SAM is converted to 5′-methylthioadenosine (MTA) as a direct methyl donor. In this study, we report identification of several nucleoside-linked shunt products in the NosN-catalyzed reaction. Comparative analysis of NosN and the class A RSMT RlmN and further density functional theory (DFT) calculations reveal important mechanistic insights into the catalyses of the two types of enzymes, showing that the radical intermediates generated by similar pathways can have very diverse reactivities. This investigation provides strong evidence supporting the previous mechanistic proposal of NosN catalysis, validating the presence of a key radical adduct that results from the addition of an MTA-derived methylene radical onto the C4 of the indolyl substrate.
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substrate tuned catalysis of the radical s adenosyl l methionine enzyme nosl involved in Nosiheptide biosynthesis
Angewandte Chemie, 2015Co-Authors: Wei Ding, Qi ZhangAbstract:NosL is a radical S-adenosyl-L-methionine (SAM) enzyme that converts L-Trp to 3-methyl-2-indolic acid, a key intermediate in the biosynthesis of a thiopeptide antibiotic Nosiheptide. In this work we investigated NosL catalysis by using a series of Trp analogues as the molecular probes. Using a benzofuran substrate 2-amino-3-(benzofuran-3-yl)propanoic acid (ABPA), we clearly demonstrated that the 5'-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction in NosL catalysis is not from the indole nitrogen but likely from the amino group of L-Trp. Unexpectedly, the major product of ABPA is a decarboxylated compound, indicating that NosL was transformed to a novel decarboxylase by an unnatural substrate. Furthermore, we showed that, for the first time to our knowledge, the dAdo radical-mediated hydrogen abstraction can occur from an alcohol hydroxy group. Our study demonstrates the intriguing promiscuity of NosL catalysis and highlights the potential of engineering radical SAM enzymes for novel activities.
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radical mediated enzymatic carbon chain fragmentation recombination
Nature Chemical Biology, 2011Co-Authors: Qi Zhang, Lian Duan, Ben Shen, Dandan Chen, Wen LiuAbstract:The radical S-adenosylmethionine (S-AdoMet) superfamily contains thousands of proteins that catalyze highly diverse conversions, most of which are poorly understood, owing to a lack of information regarding chemical products and radical-dependent transformations. We here report that NosL, involved in forming the indole side ring of the thiopeptide Nosiheptide (NOS), is a radical S-AdoMet 3-methyl-2-indolic acid (MIA) synthase. NosL catalyzed an unprecedented carbon chain reconstitution of L-tryptophan to give MIA, showing removal of the Cα-N unit and shift of the carboxylate to the indole ring. Dissection of the enzymatic process upon the identification of products and a putative glycyl intermediate uncovered a radical-mediated, unusual fragmentation-recombination reaction. This finding unveiled a key step in radical S-AdoMet enzyme-catalyzed structural rearrangements during complex biotransformations. Additionally, NosL tolerated fluorinated L-tryptophan as the substrate, allowing for production of a regiospecifically halogenated thiopeptide that has not been found among the more than 80 members of the naturally occurring thiopeptide family.
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nosa catalyzing carboxyl terminal amide formation in Nosiheptide maturation via an enamine dealkylation on the serine extended precursor peptide
Journal of the American Chemical Society, 2010Co-Authors: Heng Guo, Qi Zhang, Lian Duan, Ying Ding, Rijing Liao, Chun Lei, Ben Shen, Wen LiuAbstract:The carboxyl-terminal amide group has been often found in many bioactive peptide natural products, including Nosiheptide belonging to the over 80 entity-containing thiopeptide family. Upon functional characterization of a novel protein NosA in Nosiheptide biosynthesis, herein we report an unusual C-terminal amide forming strategy in general for maturating certain amide-terminated thiopeptides by processing their precursor peptides featuring a serine extension. NosA acts on an intermediate bearing a bis-dehydroalanine tail and catalyzes an enamide dealkylation to remove the acrylate unit originating from the extended serine residue.
