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Carmen Méndez - One of the best experts on this subject based on the ideXlab platform.
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Chapter 11. Sugar biosynthesis and modification.
Methods in Enzymology, 2009Co-Authors: Felipe Lombó, Carlos Olano, José A. Salas, Carmen MéndezAbstract:Abstract Many bioactive compounds contain as part of their molecules one or more deoxysugar units. Their presence in the final compound is generally necessary for biological activity. These sugars derive from common monosaccharides, like d ‐glucose, which have lost one or more hydroxyl groups (monoDeoxysugars, diDeoxysugars, triDeoxysugars) during their biosynthesis. These Deoxysugars are transferred to the final molecule by the action of a glycosyltransferase. Here, we first summarize the different biosynthetic steps required for the generation of the different families of Deoxysugars, including those containing extra methyl or amino groups, or tailoring modifications of the glycosylated compounds. We then give examples of several strategies for modification of the glycosylation pattern of a given bioactive compound: inactivation of genes involved in the biosynthesis of Deoxysugars; heterologous expression of genes for the biosynthesis or transfer of a specific deoxysugar; and combinatorial biosynthesis (including the use of gene cassette plasmids). Finally, we report techniques for the isolation and detection of the new glycosylated derivatives generated using these strategies.
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modulation of deoxysugar transfer by the elloramycin glycosyltransferase elmgt through site directed mutagenesis
Journal of Bacteriology, 2009Co-Authors: Angelina Ramos, Carlos Olano, Carmen Méndez, Alejandro F. Braña, José A. SalasAbstract:The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different l- and d-Deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the α/β/α motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific Deoxysugars.
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combinatorial biosynthesis of antitumor deoxysugar pathways in streptomyces griseus reconstitution of unnatural natural gene clusters for the biosynthesis of four 2 6 d dideoxyhexoses
Applied and Environmental Microbiology, 2006Co-Authors: María S. Pérez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Irfan Baig, Carmen MéndezAbstract:Combinatorial biosynthesis was applied to Streptomyces deoxysugar biosynthesis genes in order to reconstitute “unnatural natural gene clusters” for the biosynthesis of four d-Deoxysugars (d-olivose, d-oliose, d-digitoxose, and d-boivinose). Expression of these gene clusters in Streptomyces albus 16F4 was used to prove the functionality of the designed clusters through the generation of glycosylated tetracenomycins. Three glycosylated tetracenomycins were generated and characterized, two of which (d-digitoxosyl-tetracenomycin C and d-boivinosyl-tetracenocmycin C) were novel compounds. The constructed gene clusters may be used to increase the capabilities of microorganisms to synthesize new Deoxysugars and therefore to produce new glycosylated bioactive compounds.
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Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes.
Applied Microbiology and Biotechnology, 2006Co-Authors: Leandro M Garrido, Felipe Lombó, José A. Salas, Carmen Méndez, Alejandro F. Braña, Irfan Baig, Mohammad Nur-e-alam, Renata L. A. Furlan, Charlotte C. Borda, Jurgen RohrAbstract:Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring Deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.
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Metabolic engineering of the heterologous production of clorobiocin derivatives and elloramycin in Streptomyces coelicolor M512.
Metabolic Engineering, 2006Co-Authors: Anja Freitag, José A. Salas, Carmen Méndez, Bernd Kammerer, Lutz HeideAbstract:Abstract The aminocoumarin antibiotic clorobiocin is a potent inhibitor of bacterial gyrase. Two new analogs of clorobiocin could be obtained by deletion of a methyltransferase gene, involved in deoxysugar biosynthesis, from the biosynthetic gene cluster of clorobiocin, followed by expression of the modified cluster in the heterologous host Streptomyces coelicolor M512. However, only low amounts of the desired glycosides were formed, and aminocoumarins accumulated predominantly in form of aglyca. In the present study, we clarified the limiting steps for aminocoumarin glycoside formation, and devised strategies to improve glycosylation efficiency. Heterologous expression of a partial elloramycin biosynthetic gene cluster indicated that the rate of dTDP- l -rhamnose synthesis, rather than the rate of glycosyl transfer, was limiting for glycoside formation in this strain. Introduction of plasmid pRHAM which contains four genes from the oleandomycin biosynthetic gene cluster, directing the synthesis of dTDP-rhamnose, led to a 26-fold increase of the production of glycosylated aminocoumarins. Expression of the 4-ketoreductase gene oleU alone resulted in an 8-fold increase. Structural investigation of the resulting Deoxysugars confirmed that both the endogeneous and the heterologous pathway involve a 3,5-epimerization of the deoxysugar, a hypothesis which had recently been questioned.
José A. Salas - One of the best experts on this subject based on the ideXlab platform.
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formation and attachment of the deoxysugar moiety and assembly of the gene cluster for caprazamycin biosynthesis
Applied and Environmental Microbiology, 2010Co-Authors: Leonard Kaysser, José A. Salas, Emmanuel Wemakor, Stefanie Siebenberg, Bernd Kammerer, Jae Kyung Sohng, Bertolt GustAbstract:Caprazamycins are potent antimycobacterials isolated from Streptomyces sp. MK730F-62F2 (23). In a pulmonary tuberculosis mouse model, they showed a therapeutic effect but no significant toxicity (24). The caprazamycins are assigned to the translocase I inhibitors (27) due to their structural similarity to the liposidomycins (34, 43), which have been studied in more detail. Translocase I catalyzes the first step in the membrane-linked reaction cycle of bacterial cell wall formation (52): the transfer of phospho-N-acetylmuramic acid-l-Ala-γ-d-Glu-m-diaminopimelic acid-d-Ala-d-Ala from UMP to the lipid carrier undecaprenyl phosphate. Structurally unique in nature, the caprazamycins and liposidomycins share a 5′-β-O-aminoribosyl-glycyluridine and a rare N-methylated diazepanone as their characteristic feature (Fig. (Fig.1)1) (25, 53). Attached at the 3″ position are β-hydroxylated fatty acid groups of different chain lengths, carrying a 3-methylglutarate. While the liposidomycins are sulfated, the caprazamycins lack this group. Instead, they are glycosylated with a 2,3,4-O-methyl-l-rhamnose and therefore belong to the large number of bioactive compounds containing 6-deoxyhexoses. Usually, these moieties contribute significantly to the compounds' properties, influencing, e.g., molecule-target interactions, cell import and export, pharmacokinetics, and solubility (56). The biosynthesis of Deoxysugars has been studied in detail and generally starts from NDP-activated hexoses via 4-keto-6-deoxy intermediates (36). The formation of l-rhamnose involves four enzymes, a dTDP-d-glucose synthase, a dTDP-d-glucose 4,6-dehydratase, a dTDP-3,5-deoxyglucose epimerase, and a 4-ketoreductase. Recent advances in combinatorial biosynthesis have led to a variety of novel natural products with an engineered glycosylation pattern and altered bioactivity (37, 44). The key step in this approach is the attachment of different Deoxysugars to the aglycones, which demands substrate-flexible glycosyltransferases. FIG. 1. Structure of caprazamycins and organization of the caprazamycin gene cluster (cpz9 to cpz31) lacking the genes for deoxysugar formation. Assignments of genes to different steps in the biosynthesis are indicated. We recently reported cloning and heterologous expression of cosmid cpzLK09 containing the first identified gene cluster of a translocase I inhibitor, the caprazamycins. However, genes for the formation of the dTDP-l-rhamnose could not be identified on this cosmid (Fig. (Fig.1)1) and therefore only caprazamycin aglycones were produced by the heterologous host, Streptomyces coelicolor M512/cpzLK09 (31). The absence of genes for the formation of the l-rhamnose moiety within the corresponding biosynthetic gene cluster has previously been reported for aranciamycin (38), steffimycin (16), spinosyn (55), and elloramycin (9). Since potential genes for O methylation (cpz28 to cpz30) and a glycosyltransferase (cpz31) are encoded in the caprazamycin gene cluster, we speculated that only four genes are missing for successful heterologous production of intact caprazamycins. Here we report the identification of the genes required for the biosynthesis of the caprazamycin deoxysugar moiety elsewhere on the genome of Streptomyces sp. MK730-62F2. A new strategy was developed, based on Red/ET-mediated recombination, to assemble the identified subcluster into cosmid cpzLK09. Expression of the assembled cluster readily resulted in the production of intact caprazamycins in the heterologous producer strain. Moreover, in vitro studies demonstrated that Cpz31 is the glycosyltransferase in caprazamycin biosynthesis.
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Chapter 11. Sugar biosynthesis and modification.
Methods in Enzymology, 2009Co-Authors: Felipe Lombó, Carlos Olano, José A. Salas, Carmen MéndezAbstract:Abstract Many bioactive compounds contain as part of their molecules one or more deoxysugar units. Their presence in the final compound is generally necessary for biological activity. These sugars derive from common monosaccharides, like d ‐glucose, which have lost one or more hydroxyl groups (monoDeoxysugars, diDeoxysugars, triDeoxysugars) during their biosynthesis. These Deoxysugars are transferred to the final molecule by the action of a glycosyltransferase. Here, we first summarize the different biosynthetic steps required for the generation of the different families of Deoxysugars, including those containing extra methyl or amino groups, or tailoring modifications of the glycosylated compounds. We then give examples of several strategies for modification of the glycosylation pattern of a given bioactive compound: inactivation of genes involved in the biosynthesis of Deoxysugars; heterologous expression of genes for the biosynthesis or transfer of a specific deoxysugar; and combinatorial biosynthesis (including the use of gene cassette plasmids). Finally, we report techniques for the isolation and detection of the new glycosylated derivatives generated using these strategies.
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modulation of deoxysugar transfer by the elloramycin glycosyltransferase elmgt through site directed mutagenesis
Journal of Bacteriology, 2009Co-Authors: Angelina Ramos, Carlos Olano, Carmen Méndez, Alejandro F. Braña, José A. SalasAbstract:The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different l- and d-Deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the α/β/α motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific Deoxysugars.
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combinatorial biosynthesis of antitumor deoxysugar pathways in streptomyces griseus reconstitution of unnatural natural gene clusters for the biosynthesis of four 2 6 d dideoxyhexoses
Applied and Environmental Microbiology, 2006Co-Authors: María S. Pérez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Irfan Baig, Carmen MéndezAbstract:Combinatorial biosynthesis was applied to Streptomyces deoxysugar biosynthesis genes in order to reconstitute “unnatural natural gene clusters” for the biosynthesis of four d-Deoxysugars (d-olivose, d-oliose, d-digitoxose, and d-boivinose). Expression of these gene clusters in Streptomyces albus 16F4 was used to prove the functionality of the designed clusters through the generation of glycosylated tetracenomycins. Three glycosylated tetracenomycins were generated and characterized, two of which (d-digitoxosyl-tetracenomycin C and d-boivinosyl-tetracenocmycin C) were novel compounds. The constructed gene clusters may be used to increase the capabilities of microorganisms to synthesize new Deoxysugars and therefore to produce new glycosylated bioactive compounds.
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Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes.
Applied Microbiology and Biotechnology, 2006Co-Authors: Leandro M Garrido, Felipe Lombó, José A. Salas, Carmen Méndez, Alejandro F. Braña, Irfan Baig, Mohammad Nur-e-alam, Renata L. A. Furlan, Charlotte C. Borda, Jurgen RohrAbstract:Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring Deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.
Yashwant D. Vankar - One of the best experts on this subject based on the ideXlab platform.
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One-Step TEMPO-Catalyzed and Water-Mediated Stereoselective Conversion of Glycals into 2-Azido-2-Deoxysugars with a PIFA–Trimethylsilyl Azide Reagent System
Organic Letters, 2018Co-Authors: Ande Chennaiah, Yashwant D. VankarAbstract:An unprecedented water-mediated and TEMPO-catalyzed, one-step, highly regiospecific and stereoselective functionalization of glycals to 2-azido-2-Deoxysugars has been developed using a PIFA–Me3SiN3 reagent system in the presence of Bu4NHSO4 as a phase-transfer catalyst. The method is metal-free, proceeds under mild reaction conditions, and tolerates a variety of protecting groups. Using this method, the synthesis of an important trisaccharide unit bound by the monoclonal anti-I Ma antibody has also been demonstrated.
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One-Step TEMPO-Catalyzed and Water-Mediated Stereoselective Conversion of Glycals into 2-Azido-2-Deoxysugars with a PIFA-Trimethylsilyl Azide Reagent System
Organic Letters, 2018Co-Authors: Ande Chennaiah, Yashwant D. VankarAbstract:An unprecedented water-mediated and TEMPO-catalyzed, one-step, highly regiospecific and stereoselective functionalization of glycals to 2-azido-2-Deoxysugars has been developed using a PIFA−Me 3 SiN 3 reagent system in the presence of Bu 4 NHSO 4 as a phase-transfer catalyst. The method is metal-free, proceeds under mild reaction conditions, and tolerates a variety of protecting groups. Using this method, the synthesis of an important trisaccharide unit bound by the monoclonal anti-I Ma antibody has also been demonstrated.
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One-Step TEMPO-Catalyzed and Water-Mediated Stereoselective Conversion of Glycals into 2‑Azido-2-Deoxysugars with a PIFA–Trimethylsilyl Azide Reagent System
2018Co-Authors: Ande Chennaiah, Yashwant D. VankarAbstract:An unprecedented water-mediated and TEMPO-catalyzed, one-step, highly regiospecific and stereoselective functionalization of glycals to 2-azido-2-Deoxysugars has been developed using a PIFA–Me3SiN3 reagent system in the presence of Bu4NHSO4 as a phase-transfer catalyst. The method is metal-free, proceeds under mild reaction conditions, and tolerates a variety of protecting groups. Using this method, the synthesis of an important trisaccharide unit bound by the monoclonal anti-I Ma antibody has also been demonstrated
Jurgen Rohr - One of the best experts on this subject based on the ideXlab platform.
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investigating mithramycin deoxysugar biosynthesis enzymatic total synthesis of tdp d olivose
ChemBioChem, 2011Co-Authors: Guojun Wang, Pallab Pahari, Madan K Kharel, Jurgen RohrAbstract:Natural products often possess one or more highly modified deoxysugar moieties, which are generally critical for their biological activities.[1-3] Mithramycin (MTM), an aureolic acid-type anticancer agent, contains five deoxyhexoses, a trisaccharidal chain (d-olivosyl-3-1-d-oliosyl-3-1-d-mycarosyl) and a disaccharidal chain (d-olivosyl-3-1-d-olivosyl) attached at 2- and 6-positions of the aglycon, respectively.[4,5] Among all sugars, d-olivose is the main building component (sugars A, B and C in Scheme 1). In addition to MTM, d-olivose is also an essential component of many biologically active natural products, including all other aureolic acid-family anticancer agents and many angucyclines.[5-8] Recently, increasing effort has been made to alter the deoxysugar moiety of natural products (glycodiversification), as modifications to these important structural motifs can greatly influence their activity and substrate specificity.[3,9-14] This approach requires a thorough understanding of deoxysugar biosynthesis as well as suitable glycosyltransferases (GTs), a group of enzymes able to transfer sugars to a given acceptor substrate.[15-17] However, most deoxysugar biosynthesis and glycosylation events, including those involved in MTM biosynthesis, have not been fully characterized in vitro due to the unavailability of sugar donor substrates and soluble glycosyltransferases.[2,9,18] In the MTM pathway, genes involved in the biosynthesis and attachment of Deoxysugars have largely been investigated by in vivo studies.[5,19-23] The presence of five sugar moieties but only four GTs indicated that the MTM pathway does not follow the typical “one GT one glycosylation event” rule. Specifically, a unique GT MtmGIV has been suggested to be responsible for the first and third glycosylation events (attaching sugars C and E, respectively), whereas sugars D, A and B are transferred by MtmGIII, MtmGI and MtmGII, respectively.[22-24] Here, we report an in vitro large-scale preparation of thymidine diphosphate (TDP)-d-olivose based on the investigation of several MTM deoxysugar biosynthetic enzymes.
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combinatorial biosynthesis of antitumor deoxysugar pathways in streptomyces griseus reconstitution of unnatural natural gene clusters for the biosynthesis of four 2 6 d dideoxyhexoses
Applied and Environmental Microbiology, 2006Co-Authors: María S. Pérez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Irfan Baig, Carmen MéndezAbstract:Combinatorial biosynthesis was applied to Streptomyces deoxysugar biosynthesis genes in order to reconstitute “unnatural natural gene clusters” for the biosynthesis of four d-Deoxysugars (d-olivose, d-oliose, d-digitoxose, and d-boivinose). Expression of these gene clusters in Streptomyces albus 16F4 was used to prove the functionality of the designed clusters through the generation of glycosylated tetracenomycins. Three glycosylated tetracenomycins were generated and characterized, two of which (d-digitoxosyl-tetracenomycin C and d-boivinosyl-tetracenocmycin C) were novel compounds. The constructed gene clusters may be used to increase the capabilities of microorganisms to synthesize new Deoxysugars and therefore to produce new glycosylated bioactive compounds.
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Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes.
Applied Microbiology and Biotechnology, 2006Co-Authors: Leandro M Garrido, Felipe Lombó, José A. Salas, Carmen Méndez, Alejandro F. Braña, Irfan Baig, Mohammad Nur-e-alam, Renata L. A. Furlan, Charlotte C. Borda, Jurgen RohrAbstract:Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring Deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.
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Engineering Biosynthetic Pathways for Deoxysugars: Branched-Chain Sugar Pathways and Derivatives from the Antitumor Tetracenomycin
Chemistry & Biology, 2004Co-Authors: Felipe Lombó, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Miranda Gibson, Lisa Greenwell, Carmen MéndezAbstract:Summary Sugar biosynthesis cassette genes have been used to construct plasmids directing the biosynthesis of branched-chain Deoxysugars: pFL942 (NDP-L-mycarose), pFL947 (NDP-4-deacetyl-L-chromose B), and pFL946/pFL954 (NDP-2,3,4-tridemethyl-L-nogalose). Expression of pFL942 and pFL947 in S. lividans 16F4, which harbors genes for elloramycinone biosynthesis and the flexible ElmGT glycosyltransferase of the elloramycin biosynthetic pathway, led to the formation of two compounds: 8-α-L-mycarosyl-elloramycinone and 8-demethyl-8-(4-deacetyl)-α-L-chromosyl-tetracenomycin C, respectively. Expression of pFL946 or pFL954 failed to produce detectable amounts of a novel glycosylated tetracenomycin derivative. Formation of these two compounds represents examples of the sugar cosubstrate flexibility of the ElmGT glycosyltransferase. The use of these cassette plasmids also provided insights into the substrate flexibility of deoxysugar biosynthesis enzymes as the C -methyltransferases EryBIII and MtmC, the epimerases OleL and EryBVII, and the 4-ketoreductases EryBIV and OleU.
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biosynthesis of the antitumor chromomycin a3 in streptomyces griseus analysis of the gene cluster and rational design of novel chromomycin analogs
Chemistry & Biology, 2004Co-Authors: Nuria Menendez, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Mohammad Nurealam, Carmen MéndezAbstract:Abstract The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A 3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A 3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A 3 and its Deoxysugars is proposed.
Alejandro F. Braña - One of the best experts on this subject based on the ideXlab platform.
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modulation of deoxysugar transfer by the elloramycin glycosyltransferase elmgt through site directed mutagenesis
Journal of Bacteriology, 2009Co-Authors: Angelina Ramos, Carlos Olano, Carmen Méndez, Alejandro F. Braña, José A. SalasAbstract:The glycosyltransferase ElmGT from Streptomyces olivaceus is involved in the biosynthesis of the antitumor drug elloramycin, and it has been shown to possess a broad deoxysugar recognition pattern, being able to transfer different l- and d-Deoxysugars to 8-demethyl-tetracenomycin C, the elloramycin aglycone. Site-directed mutagenesis in residues L309 and N312, located in the α/β/α motif within the nucleoside diphosphate-sugar binding region, can be used to modulate the substrate flexibility of ElmGT, making it more precise for transfer of specific Deoxysugars.
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combinatorial biosynthesis of antitumor deoxysugar pathways in streptomyces griseus reconstitution of unnatural natural gene clusters for the biosynthesis of four 2 6 d dideoxyhexoses
Applied and Environmental Microbiology, 2006Co-Authors: María S. Pérez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Irfan Baig, Carmen MéndezAbstract:Combinatorial biosynthesis was applied to Streptomyces deoxysugar biosynthesis genes in order to reconstitute “unnatural natural gene clusters” for the biosynthesis of four d-Deoxysugars (d-olivose, d-oliose, d-digitoxose, and d-boivinose). Expression of these gene clusters in Streptomyces albus 16F4 was used to prove the functionality of the designed clusters through the generation of glycosylated tetracenomycins. Three glycosylated tetracenomycins were generated and characterized, two of which (d-digitoxosyl-tetracenomycin C and d-boivinosyl-tetracenocmycin C) were novel compounds. The constructed gene clusters may be used to increase the capabilities of microorganisms to synthesize new Deoxysugars and therefore to produce new glycosylated bioactive compounds.
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Insights in the glycosylation steps during biosynthesis of the antitumor anthracycline cosmomycin: characterization of two glycosyltransferase genes.
Applied Microbiology and Biotechnology, 2006Co-Authors: Leandro M Garrido, Felipe Lombó, José A. Salas, Carmen Méndez, Alejandro F. Braña, Irfan Baig, Mohammad Nur-e-alam, Renata L. A. Furlan, Charlotte C. Borda, Jurgen RohrAbstract:Glycosylation pattern in cosmomycins is a distinctive feature among anthracyclines. These antitumor compounds possess two trisaccharide chains attached at C-7 and C-10, each of them with structural variability, mainly at the distal deoxysugar moieties. We have characterized a 14-kb chromosomal region from Streptomyces olindensis containing 13 genes involved in cosmomycin biosynthesis. Two of the genes, cosG and cosK, coding for glycosyltransferase were inactivated with the generation of five new derivatives. Structural elucidation of these compounds showed altered glycosylation patterns indicating the capability of both glycosyltransferases of transferring Deoxysugars to both sides of the aglycone and the flexibility of CosK with respect to the deoxysugar donor. A model is proposed for the glycosylation steps during cosmomycins biosynthesis.
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Engineering Biosynthetic Pathways for Deoxysugars: Branched-Chain Sugar Pathways and Derivatives from the Antitumor Tetracenomycin
Chemistry & Biology, 2004Co-Authors: Felipe Lombó, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Miranda Gibson, Lisa Greenwell, Carmen MéndezAbstract:Summary Sugar biosynthesis cassette genes have been used to construct plasmids directing the biosynthesis of branched-chain Deoxysugars: pFL942 (NDP-L-mycarose), pFL947 (NDP-4-deacetyl-L-chromose B), and pFL946/pFL954 (NDP-2,3,4-tridemethyl-L-nogalose). Expression of pFL942 and pFL947 in S. lividans 16F4, which harbors genes for elloramycinone biosynthesis and the flexible ElmGT glycosyltransferase of the elloramycin biosynthetic pathway, led to the formation of two compounds: 8-α-L-mycarosyl-elloramycinone and 8-demethyl-8-(4-deacetyl)-α-L-chromosyl-tetracenomycin C, respectively. Expression of pFL946 or pFL954 failed to produce detectable amounts of a novel glycosylated tetracenomycin derivative. Formation of these two compounds represents examples of the sugar cosubstrate flexibility of the ElmGT glycosyltransferase. The use of these cassette plasmids also provided insights into the substrate flexibility of deoxysugar biosynthesis enzymes as the C -methyltransferases EryBIII and MtmC, the epimerases OleL and EryBVII, and the 4-ketoreductases EryBIV and OleU.
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biosynthesis of the antitumor chromomycin a3 in streptomyces griseus analysis of the gene cluster and rational design of novel chromomycin analogs
Chemistry & Biology, 2004Co-Authors: Nuria Menendez, José A. Salas, Jurgen Rohr, Alejandro F. Braña, Mohammad Nurealam, Carmen MéndezAbstract:Abstract The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A 3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A 3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A 3 and its Deoxysugars is proposed.