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Ben Shen - One of the best experts on this subject based on the ideXlab platform.
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blmb and tlmb provide resistance to the bleomycin family of antitumor antibiotics by n acetylating metal free bleomycin tallysomycin phleomycin and zorbamycin
Biochemistry, 2014Co-Authors: Jane M Coughlin, Ute Galm, Evelyn Wendtpienkowski, Liyan Wang, Claudia Unsin, Jeffrey D Rudolf, Dong Yang, Meifeng Tao, Ben ShenAbstract:The bleomycin (BLM) family of glycopeptide-derived antitumor antibiotics consists of BLMs, tallysomycins (TLMs), phleomycins (PLMs), and zorbamycin (ZBM). The self-resistant elements BlmB and TlmB, discovered from the BLM- and TLM-producing organisms Streptomyces verticillus ATCC15003 and Streptoalloteichus hindustanus E465-94 ATCC31158, respectively, are N-acetyltransferases that provide resistance to the producers by disrupting the metal-binding domain of the antibiotics required for activity. Although each member of the BLM family of antibiotics possesses a conserved metal-binding domain, the structural differences between each member, namely, the bithiazole moiety and C-terminal amine of BLMs, have been suggested to instill substrate specificity within BlmB. Here we report that BlmB and TlmB readily accept and acetylate BLMs, TLMs, PLMs, and ZBM in vitro but only in the metal-free forms. Kinetic analysis of BlmB and TlmB reveals there is no strong preference or rate enhancement for specific substrates...
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the crystal structure of blmi as a model for nonribosomal peptide synthetase peptidyl carrier proteins
Proteins, 2014Co-Authors: Jeremy R Lohman, Marianne E Cuff, Lance Bigelow, Jessica Bearden, Gyorgy Babnigg, Andrzej Joachimiak, George N Phillips, Ben ShenAbstract:Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.
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A Designer Bleomycin with Significantly Improved DNA Cleavage Activity
2012Co-Authors: Shengxiong Huang, Ute Galm, Liyan Wang, Jane M Coughlin, Dong Yang, Meifeng Tao, Zhiyang Feng, Evelyn Wendt-pienkowski, Yanwen Duan, Ben ShenAbstract:The bleomycins (BLMs) are used clinically in combination with a number of other agents for the treatment of several types of tumors, and the BLM, etoposide, and cisplatin treatment regimen cures 90–95% of metastatic testicular cancer patients. BLM-induced pneumonitis is the most feared, dose-limiting side effect of BLM in chemotherapy, which can progress into lung fibrosis and affect up to 46% of the total patient population. There have been continued efforts to develop new BLM analogues in the search for anticancer drugs with better clinical efficacy and lower lung toxicity. We have previously cloned and characterized the biosynthetic gene clusters for BLMs from Streptomyces verticillus ATCC15003, tallysomycins from Streptoalloteichus hindustanus E465-94 ATCC31158, and zorbamycin (ZBM) from Streptomyces flavoviridis SB9001. Comparative analysis of the three biosynthetic machineries provided the molecular basis for the formulation of hypotheses to engineer novel analogues. We now report engineered production of three new analogues, 6′-hydroxy-ZBM, BLM Z, and 6′-deoxy-BLM Z and the evaluation of their DNA cleavage activities as a measurement for their potential anticancer activity. Our findings unveiled: (i) the disaccharide moiety plays an important role in the DNA cleavage activity of BLMs and ZBMs, (ii) the ZBM disaccharide significantly enhances the potency of BLM, and (iii) 6′-deoxy-BLM Z represents the most potent BLM analogue known to date. The fact that 6′-deoxy-BLM Z can be produced in reasonable quantities by microbial fermentation should greatly facilitate follow-up mechanistic and preclinical studies to potentially advance this analogue into a clinical drug
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comparative analysis of the biosynthetic gene clusters and pathways for three structurally related antitumor antibiotics bleomycin tallysomycin and zorbamycin
Journal of Natural Products, 2011Co-Authors: Ute Galm, Evelyn Wendtpienkowski, Liyan Wang, Shengxiong Huang, Claudia Unsin, Jane M Coughlin, Ben ShenAbstract:The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and contrast their respective biosynthetic pathways and to investigate various enzymatic elements. The presence of different numbers of isolated nonribosomal peptide synthetase (NRPS) domains in all three clusters does not result in major structural differences of the respective compounds. The seemingly identical domain organization of the NRPS modules responsible for heterocycle formation, on the other hand, is contrasted by the biosynthesis of two different structural entities, bithiazole and thiazolinyl-thiazole, for ...
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the tallysomycin biosynthetic gene cluster from streptoalloteichus hindustanus e465 94 atcc 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics
Molecular BioSystems, 2007Co-Authors: Liyan Wang, Ute Galm, Evelyn Wendtpienkowski, Jane M Coughlin, Nicholas P George, Guodong Zhang, Ben ShenAbstract:The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmEglycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the ΔtlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.
Liangcheng Du - One of the best experts on this subject based on the ideXlab platform.
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the biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus atcc15003 as a model for hybrid peptide polyketide natural product biosynthesis
Journal of Industrial Microbiology & Biotechnology, 2001Co-Authors: Ben Shen, Liangcheng Du, Cesar Sanchez, Daniel J Edwards, Mei Chen, Jeffrey M MurrellAbstract:The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3) posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385.
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the biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus atcc15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase
Chemistry & Biology, 2000Co-Authors: Liangcheng Du, Cesar Sanchez, Daniel J Edwards, Mei Chen, Ben ShenAbstract:Background: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS^PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide^polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. Results: A set of 30 genes clustered with the previously characterized blmAB resistance genes were de¢ned by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, ¢ve sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate speci¢cities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid speci¢cities of two NRPS modules were con¢rmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. Conclusion: The blm gene cluster is characterized by a hybrid NRPS^PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide^polyketide metabolites.
Alejandro D Bolzan - One of the best experts on this subject based on the ideXlab platform.
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dna and chromosome damage induced by bleomycin in mammalian cells an update
Mutation Research-reviews in Mutation Research, 2018Co-Authors: Alejandro D Bolzan, Martha S BianchiAbstract:Bleomycin (BLM) is an antibiotic isolated from Streptomyces verticillus. It has radiomimetic actions on DNA thus it has been widely used in clinical chemotherapy for the treatment of different types of cancer, including head and neck tumors, lymphomas, squamous-cell carcinomas and germ-cell tumors. Because of this, the study of BLM genotoxicity is of practical interest. This antibiotic is an S-independent clastogen and an agent that generates free radicals and induces single- and double-strand breaks in DNA. In the present review, we will summarize our current knowledge concerning the DNA and chromosome damage induced by BLM in mammalian cells, with emphasis on new developments published since 1991.
Mei Chen - One of the best experts on this subject based on the ideXlab platform.
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the biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus atcc15003 as a model for hybrid peptide polyketide natural product biosynthesis
Journal of Industrial Microbiology & Biotechnology, 2001Co-Authors: Ben Shen, Liangcheng Du, Cesar Sanchez, Daniel J Edwards, Mei Chen, Jeffrey M MurrellAbstract:The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3) posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385.
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the biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus atcc15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase
Chemistry & Biology, 2000Co-Authors: Liangcheng Du, Cesar Sanchez, Daniel J Edwards, Mei Chen, Ben ShenAbstract:Background: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS^PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide^polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. Results: A set of 30 genes clustered with the previously characterized blmAB resistance genes were de¢ned by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, ¢ve sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate speci¢cities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid speci¢cities of two NRPS modules were con¢rmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. Conclusion: The blm gene cluster is characterized by a hybrid NRPS^PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide^polyketide metabolites.
Daniel J Edwards - One of the best experts on this subject based on the ideXlab platform.
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the biosynthetic gene cluster for the anticancer drug bleomycin from Streptomyces verticillus atcc15003 as a model for hybrid peptide polyketide natural product biosynthesis
Journal of Industrial Microbiology & Biotechnology, 2001Co-Authors: Ben Shen, Liangcheng Du, Cesar Sanchez, Daniel J Edwards, Mei Chen, Jeffrey M MurrellAbstract:The hybrid peptide–polyketide backbone of bleomycin (BLM) is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules. BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems. Sequence analysis and functional comparison of domains and modules of BlmIX/BlmVIII/BlmVII with those of nonhybrid NRPS and PKS systems suggest that (1) the same catalytic sites appear to be conserved in both hybrid NRPS–PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (2) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate transfer of the growing intermediates between the interacting NRPS and/or PKS modules; and (3) posttranslational modification of the BLM megasynthetase has been accomplished by a single PPTase with a broad substrate specificity toward the apo forms of both acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). Journal of Industrial Microbiology & Biotechnology (2001) 27, 378–385.
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the biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus atcc15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase
Chemistry & Biology, 2000Co-Authors: Liangcheng Du, Cesar Sanchez, Daniel J Edwards, Mei Chen, Ben ShenAbstract:Background: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS^PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide^polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. Results: A set of 30 genes clustered with the previously characterized blmAB resistance genes were de¢ned by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, ¢ve sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate speci¢cities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid speci¢cities of two NRPS modules were con¢rmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. Conclusion: The blm gene cluster is characterized by a hybrid NRPS^PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide^polyketide metabolites.