The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Christian Hertweck - One of the best experts on this subject based on the ideXlab platform.
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Electrochemical monitoring of ROS generation by anticancer agents: the case of Chartreusin
RSC Advances, 2017Co-Authors: Antonio Doménech-carbó, Nico Ueberschaar, Gerardo Cebrían-torrejón, Noemínoemí Montoya, Marcus Tullius Scotti, Zohra Benfodda, Christian HertweckAbstract:Solution phase and solid-sate electrochemical techniques centered in the voltammetry of microparticles approach are applied for testing the cytotoxic activity of anticancer drugs. The possibility of electrochemical generation of reactive oxygen species (ROS) is exploited for evaluating their contribution to cellular damage. The described methodology is applied to the case of Chartreusin (Ch) whose electrochemistry in non-aqueous solutions and in the solid state in contact with aqueous electrolytes is described in the absence (experimental data were confirmed by theoretical calculations) and in the presence of double-stranded DNA (dsDNA). In parallel, scanning electrochemical microscopy (SECM) examination of dsDNA fibers was developed. Electrochemical data suggest that Ch-induced dsDNA interaction can operate by a two pathways: via intercalation and by mechanisms related with ROS generation. Although reduced Ch forms electrochemically generated can act as radical scavengers blocking the ROS generation chain, this property is interrupted upon binding to dsDNA.
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Bipiperidine conjugates as soluble sugar surrogates in DNA-intercalating antiproliferative polyketides.
Chemical communications (Cambridge England), 2016Co-Authors: Nico Ueberschaar, Hans-martin Dahse, Florian Meyer, Christian HertweckAbstract:DNA-intercalating polyketide glycosides are important leads for cancer therapeutics, yet their use is often limited by their low solubility and challenging synthetic protocols. To overcome these limitations, we employed 1,4′-bipiperidine-1′-carbamate residues as sugar surrogates in daunorubicin and Chartreusin, yielding water-soluble derivatives and prodrugs with dramatically improved antiproliferative activities.
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Synthetic remodeling of the Chartreusin pathway to tune antiproliferative and antibacterial activities.
Journal of the American Chemical Society, 2013Co-Authors: Nico Ueberschaar, Kirstin Scherlach, Mikko Metsä-ketelä, Tom Bretschneider, Hans-martin Dahse, Helmar Görls, Christian HertweckAbstract:Natural products of the benzonaphthopyranone class, such as Chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of Chartreusin derivatives. Pathway engineering of the Chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of Chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new Chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-Chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of...
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Rational Design of an Apoptosis-Inducing Photoreactive DNA Intercalator**
Angewandte Chemie (International ed. in English), 2013Co-Authors: Nico Ueberschaar, Tom Bretschneider, Hans-martin Dahse, Christian HertweckAbstract:Herein we report the first successful tailoring of thepotent chartarin pharmacophore by merging the strengths ofmolecular modeling, biosynthesis, and chemical synthesis. Wedemonstrate a viable chemobiosynthetic route to a novelvinyl-substituted Chartreusin analogue, which forms covalentlinkstoDNAuponmildphotoactivationwithvisiblelight andwhich has a markedly higher antitumoral potency than theparent compound.To rationally design Chartreusin analogues with poten-tially improved potencies we modeled the structures ofChartreusin and ring-substituted analogues into DNA andtookintoaccountcurrentdataonthemultivalent,nonrandombinding properties. Since Chartreusin preferentially binds tosequences containing CpG or TpG triplets,
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Synthetic Remodeling of the Chartreusin Pathway to Tune Antiproliferative and Antibacterial Activities
2013Co-Authors: Nico Ueberschaar, Kirstin Scherlach, Tom Bretschneider, Hans-martin Dahse, Mikko Metsä-ketelä, Helmar Görls, Christian HertweckAbstract:Natural products of the benzonaphthopyranone class, such as Chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of Chartreusin derivatives. Pathway engineering of the Chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of Chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new Chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-Chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of vinyl- and ethynyl-substituted derivatives with blue light resulted in an improved antiproliferative potency against a colorectal cancer cell line. In contrast, the replacement of a methyl group by hydrogen caused a drastically decreased cytotoxicity but markedly enhanced antimycobacterial activity. Furthermore, mutasynthesis of bromoChartreusin led to the first crystal structure of a Chartreusin derivative that is not modified in the glycoside residue. Beyond showcasing the possibility of converting diverse, fully synthetic polyphenolic aglycones into the corresponding glycosides in a whole-cell approach, this work identified new Chartreusins with fine-tuned properties as promising candidates for further development as therapeutics
Nico Ueberschaar - One of the best experts on this subject based on the ideXlab platform.
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Electrochemical monitoring of ROS generation by anticancer agents: the case of Chartreusin
RSC Advances, 2017Co-Authors: Antonio Doménech-carbó, Nico Ueberschaar, Gerardo Cebrían-torrejón, Noemínoemí Montoya, Marcus Tullius Scotti, Zohra Benfodda, Christian HertweckAbstract:Solution phase and solid-sate electrochemical techniques centered in the voltammetry of microparticles approach are applied for testing the cytotoxic activity of anticancer drugs. The possibility of electrochemical generation of reactive oxygen species (ROS) is exploited for evaluating their contribution to cellular damage. The described methodology is applied to the case of Chartreusin (Ch) whose electrochemistry in non-aqueous solutions and in the solid state in contact with aqueous electrolytes is described in the absence (experimental data were confirmed by theoretical calculations) and in the presence of double-stranded DNA (dsDNA). In parallel, scanning electrochemical microscopy (SECM) examination of dsDNA fibers was developed. Electrochemical data suggest that Ch-induced dsDNA interaction can operate by a two pathways: via intercalation and by mechanisms related with ROS generation. Although reduced Ch forms electrochemically generated can act as radical scavengers blocking the ROS generation chain, this property is interrupted upon binding to dsDNA.
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Bipiperidine conjugates as soluble sugar surrogates in DNA-intercalating antiproliferative polyketides.
Chemical communications (Cambridge England), 2016Co-Authors: Nico Ueberschaar, Hans-martin Dahse, Florian Meyer, Christian HertweckAbstract:DNA-intercalating polyketide glycosides are important leads for cancer therapeutics, yet their use is often limited by their low solubility and challenging synthetic protocols. To overcome these limitations, we employed 1,4′-bipiperidine-1′-carbamate residues as sugar surrogates in daunorubicin and Chartreusin, yielding water-soluble derivatives and prodrugs with dramatically improved antiproliferative activities.
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Synthetic remodeling of the Chartreusin pathway to tune antiproliferative and antibacterial activities.
Journal of the American Chemical Society, 2013Co-Authors: Nico Ueberschaar, Kirstin Scherlach, Mikko Metsä-ketelä, Tom Bretschneider, Hans-martin Dahse, Helmar Görls, Christian HertweckAbstract:Natural products of the benzonaphthopyranone class, such as Chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of Chartreusin derivatives. Pathway engineering of the Chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of Chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new Chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-Chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of...
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Rational Design of an Apoptosis-Inducing Photoreactive DNA Intercalator**
Angewandte Chemie (International ed. in English), 2013Co-Authors: Nico Ueberschaar, Tom Bretschneider, Hans-martin Dahse, Christian HertweckAbstract:Herein we report the first successful tailoring of thepotent chartarin pharmacophore by merging the strengths ofmolecular modeling, biosynthesis, and chemical synthesis. Wedemonstrate a viable chemobiosynthetic route to a novelvinyl-substituted Chartreusin analogue, which forms covalentlinkstoDNAuponmildphotoactivationwithvisiblelight andwhich has a markedly higher antitumoral potency than theparent compound.To rationally design Chartreusin analogues with poten-tially improved potencies we modeled the structures ofChartreusin and ring-substituted analogues into DNA andtookintoaccountcurrentdataonthemultivalent,nonrandombinding properties. Since Chartreusin preferentially binds tosequences containing CpG or TpG triplets,
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Synthetic Remodeling of the Chartreusin Pathway to Tune Antiproliferative and Antibacterial Activities
2013Co-Authors: Nico Ueberschaar, Kirstin Scherlach, Tom Bretschneider, Hans-martin Dahse, Mikko Metsä-ketelä, Helmar Görls, Christian HertweckAbstract:Natural products of the benzonaphthopyranone class, such as Chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of Chartreusin derivatives. Pathway engineering of the Chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of Chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new Chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-Chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of vinyl- and ethynyl-substituted derivatives with blue light resulted in an improved antiproliferative potency against a colorectal cancer cell line. In contrast, the replacement of a methyl group by hydrogen caused a drastically decreased cytotoxicity but markedly enhanced antimycobacterial activity. Furthermore, mutasynthesis of bromoChartreusin led to the first crystal structure of a Chartreusin derivative that is not modified in the glycoside residue. Beyond showcasing the possibility of converting diverse, fully synthetic polyphenolic aglycones into the corresponding glycosides in a whole-cell approach, this work identified new Chartreusins with fine-tuned properties as promising candidates for further development as therapeutics
Jose Portugal - One of the best experts on this subject based on the ideXlab platform.
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Elsamicin A binding to DNA. A comparative thermodynamic characterization.
FEBS Letters, 2004Co-Authors: Francisca Barceló, Jose PortugalAbstract:The antitumor drug elsamicin A contains a coumarin- related chartarin chromophore that intercalates into DNA. It differs from other related molecules in its disaccharide moiety, which bears an amino sugar. Its binding to DNA was analyzed using isothermal titration calorimetry and UV thermal denatur- ation, and characterized thermodynamically. For the association of elsamicin A with DNA we found DG � ¼� 8:6 kcal mol � 1 , DH ¼� 10:4 kcal mol � 1 , DS ¼� 6:1 cal mol � 1 K � 1 , and K obs ¼ 2:8ð%0:2 Þ� 10 6 M � 1 at 20 Ci n 18 mM Na þ . The contributions to the free energy of binding that lead to the DNA- elsamicin complex are compared with the binding to DNA of Chartreusin, another chartarin-containing drug. The results are discussed in terms of the contributions of the disaccharide moieties into the strength of binding. 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Elsamicin A binding to DNA. A comparative thermodynamic characterization.
FEBS letters, 2004Co-Authors: Francisca Barceló, Jose PortugalAbstract:The antitumor drug elsamicin A contains a coumarin-related chartarin chromophore that intercalates into DNA. It differs from other related molecules in its disaccharide moiety, which bears an amino sugar. Its binding to DNA was analyzed using isothermal titration calorimetry and UV thermal denaturation, and characterized thermodynamically. For the association of elsamicin A with DNA we found DeltaG degrees = -8.6 kcal mol(-1), DeltaH = -10.4 kcal mol(-1), DeltaS = -6.1 cal mol(-1) K(-1), and Kobs = 2.8(+/- 0.2) x 10(6) M(-1) at 20 degrees C in 18 mM Na+. The contributions to the free energy of binding that lead to the DNA-elsamicin complex are compared with the binding to DNA of Chartreusin, another chartarin-containing drug. The results are discussed in terms of the contributions of the disaccharide moieties into the strength of binding.
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Chartreusin, elsamicin A and related anti-cancer antibiotics.
Current medicinal chemistry. Anti-cancer agents, 2003Co-Authors: Jose PortugalAbstract:Chartreusin and elsamicin A are structurally related antibiotics that bind to GC-rich tracts in DNA, with a clear preference for B-DNA over Z-DNA. They inhibit RNA synthesis and cause single-strand scission of DNA via the formation of free radicals. Elsamicin A can also be regarded as the most potent inhibitor of topoisomerase II reported so far. It can inhibit the formation of several DNA-protein complexes. Elsamicin A binding to the P1 and P2 promoter regions of the c-myc oncogene inhibits the binding of the Sp1 transcription factor, thus inhibiting transcription. Despite the pharmacological interest in Chartreusin, elsamicin A and their derivatives, there is no experimental data on the structure of their complexes with DNA. This shortcoming has been partially solved by a theoretical approach, which provided some details about the DNA-elsamicin A interaction, and the thermodynamic characterization of the binding of Chartreusin and elsamicin A to DNA. Elsamicin A but not Chartreusin is being developed clinically as an anti-cancer agent. IST-622 (6-O-(3-ethoxypropylonyl)-3',4'-O-exo-benzylidene-Chartreusin), a novel semi-synthetic derivative of Chartreusin, which has shown a promising anti-cancer activity in a phase II study, appears to be a pro-drug with a more suitable pharmacokinetic profile than Chartreusin.
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Thermodynamic characterization of the multivalent binding of Chartreusin to DNA.
Nucleic acids research, 2002Co-Authors: Francisca Barceló, Damiana Capó, Jose PortugalAbstract:Characterization of the thermodynamics of DNA- drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic Chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 x 10(5) M(-1) at 20 degrees C, in a solution containing 18 mM Na(+). The DNA-drug interaction was enthalpy driven, with a DeltaH(b) of -7.07 kcal/mol at 20 degrees C. Binding enthalpies were determined by ITC in the 20-35 degrees C range and used to calculate a binding-induced change in heat capacity (DeltaCp) of -391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of DeltaG(obs) into the component free energy terms. The hydrophobic transfer of the Chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.
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Map of Chartreusin and elsamicin binding sites on DNA.
FEBS letters, 1991Co-Authors: Xavier Salas, Jose PortugalAbstract:Abstract Three DNA restriction fragments designated tyrT, 102-mer and 70-mer, have been used as substrates for footprinting studies using DNase I in the presence of the structurally similar antibiotics Chartreusin and elsamicin A, The sequence-selective binding sites of the antibiotics can be mapped in regions which are rich In guanine+cytosine. Chartreusin and elsamicin appear to recognize and bind preferentially to sequences containing a CpG step. Regions containing a TpG step also seem to be a good binding site. The binding of elsamicin to these sites appears to be more concentration-dependent. A comparative analysis is performed of the sizes and locations of the different binding sites, aimed to infer whether the different biological effects of Chartreusin and elsamicin A can be correlated to differences in their sequence-selective binding to DNA.
Hans-martin Dahse - One of the best experts on this subject based on the ideXlab platform.
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Bipiperidine conjugates as soluble sugar surrogates in DNA-intercalating antiproliferative polyketides.
Chemical communications (Cambridge England), 2016Co-Authors: Nico Ueberschaar, Hans-martin Dahse, Florian Meyer, Christian HertweckAbstract:DNA-intercalating polyketide glycosides are important leads for cancer therapeutics, yet their use is often limited by their low solubility and challenging synthetic protocols. To overcome these limitations, we employed 1,4′-bipiperidine-1′-carbamate residues as sugar surrogates in daunorubicin and Chartreusin, yielding water-soluble derivatives and prodrugs with dramatically improved antiproliferative activities.
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Synthetic remodeling of the Chartreusin pathway to tune antiproliferative and antibacterial activities.
Journal of the American Chemical Society, 2013Co-Authors: Nico Ueberschaar, Kirstin Scherlach, Mikko Metsä-ketelä, Tom Bretschneider, Hans-martin Dahse, Helmar Görls, Christian HertweckAbstract:Natural products of the benzonaphthopyranone class, such as Chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of Chartreusin derivatives. Pathway engineering of the Chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of Chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new Chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-Chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of...
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Rational Design of an Apoptosis-Inducing Photoreactive DNA Intercalator**
Angewandte Chemie (International ed. in English), 2013Co-Authors: Nico Ueberschaar, Tom Bretschneider, Hans-martin Dahse, Christian HertweckAbstract:Herein we report the first successful tailoring of thepotent chartarin pharmacophore by merging the strengths ofmolecular modeling, biosynthesis, and chemical synthesis. Wedemonstrate a viable chemobiosynthetic route to a novelvinyl-substituted Chartreusin analogue, which forms covalentlinkstoDNAuponmildphotoactivationwithvisiblelight andwhich has a markedly higher antitumoral potency than theparent compound.To rationally design Chartreusin analogues with poten-tially improved potencies we modeled the structures ofChartreusin and ring-substituted analogues into DNA andtookintoaccountcurrentdataonthemultivalent,nonrandombinding properties. Since Chartreusin preferentially binds tosequences containing CpG or TpG triplets,
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Synthetic Remodeling of the Chartreusin Pathway to Tune Antiproliferative and Antibacterial Activities
2013Co-Authors: Nico Ueberschaar, Kirstin Scherlach, Tom Bretschneider, Hans-martin Dahse, Mikko Metsä-ketelä, Helmar Görls, Christian HertweckAbstract:Natural products of the benzonaphthopyranone class, such as Chartreusin, elsamicin A, gilvocarcin, and polycarcin, represent potent leads for urgently needed anticancer therapeutics and antibiotics. Since synthetic protocols for altering their architectures are limited, we harnessed enzymatic promiscuity to generate a focused library of Chartreusin derivatives. Pathway engineering of the Chartreusin polyketide synthase, mutational synthesis, and molecular modeling were employed to successfully tailor the structure of Chartreusin. For the synthesis of the aglycones, improved synthetic avenues to substituted coumarin building blocks were established. Using an engineered mutant, in total 11 new Chartreusin analogs (desmethyl, methyl, ethyl, vinyl, ethynyl, bromo, hydroxy, methoxy, and corresponding (1→2) abeo-Chartreusins) were generated and fully characterized. Their biological evaluation revealed an unexpected impact of the ring substituents on antiproliferative and antibacterial activities. Irradiation of vinyl- and ethynyl-substituted derivatives with blue light resulted in an improved antiproliferative potency against a colorectal cancer cell line. In contrast, the replacement of a methyl group by hydrogen caused a drastically decreased cytotoxicity but markedly enhanced antimycobacterial activity. Furthermore, mutasynthesis of bromoChartreusin led to the first crystal structure of a Chartreusin derivative that is not modified in the glycoside residue. Beyond showcasing the possibility of converting diverse, fully synthetic polyphenolic aglycones into the corresponding glycosides in a whole-cell approach, this work identified new Chartreusins with fine-tuned properties as promising candidates for further development as therapeutics
Zhijun Wang - One of the best experts on this subject based on the ideXlab platform.
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cezomycin is activated by calc to its ester form for further biosynthesis steps in the production of calcimycin in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Hao Wu, Jingdan Liang, Xiufen Zhou, Zixin Deng, Jialiang Wang, Weijun Liang, Qiulin Wu, Ian J Bruce, Zhijun WangAbstract:ABSTRACT Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal− (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal−S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein9s apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min−1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin9s biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues9, divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin9s production.
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recycling of overactivated acyls by a type ii thioesterase during calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Jingdan Liang, Lixia Gou, Xiufen Zhou, Zixin Deng, Weijun Liang, Ian J Bruce, Zhijun WangAbstract:Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
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a novel tetr family transcriptional regulator calr3 negatively controls calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Frontiers in Microbiology, 2017Co-Authors: Lixia Gou, Tiesheng Han, Xiaoxia Wang, Wenxiu Liu, Zhijun WangAbstract:Calcimycin is a unique ionophoric antibiotic that is widely used in biochemical and pharmaceutical applications, but the genetic basis underlying the regulatory mechanisms of calcimycin biosynthesis are unclear. Here, we identified the calR3 gene, which encodes a novel TetR family transcriptional regulator and exerts a negative effect on calcimycin biosynthesis. Disruption of calR3 in Streptomyces chartreusis NRRL 3882 led to significantly increased calcimycin and its intermediate cezomycin. Gene expression analysis showed that the transcription of calR3 and its adjacent calT gene were dramatically enhanced (30- and 171-fold, respectively) in GLX26 (ΔcalR3) mutants compared with the wild-type strains. Two CalR3-binding sites within the bidirectional calR3-calT promoter region were identified using a DNase I footprinting assay, indicating that CalR3 directly repressed the transcription of its own gene and the calT gene. In vitro electrophoretic mobility shift assays suggested that both calcimycin and cezomycin can act as CalR3 ligands to induce CalR3 to dissociate from its binding sites. These findings indicate negative feedback for the regulation of CalR3 in calcimycin biosynthesis and suggest that calcimycin production can be improved by manipulating its biosynthetic machinery.
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characterization of the n methyltransferase calm involved in calcimycin biosynthesis by streptomyces chartreusis nrrl 3882
Biochimie, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Jun Yin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.
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characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic calcimycin a23187 in streptomyces chartreusis nrrl 3882
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Jingdan Liang, Shuangjun Lin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.