The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
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.
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, Alfredo F Brana, 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: Maria S Perez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alfredo F Brana, 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, Alfredo F Brana, 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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Deoxysugar transfer during chromomycin a3 biosynthesis in streptomyces griseus subsp griseus new derivatives with antitumor activity
Applied and Environmental Microbiology, 2006Co-Authors: Nuria Menendez, José A. Salas, Jurgen Rohr, Alfredo F Brana, Mohammad Nurealam, Carsten Fischer, Carmen MéndezAbstract:Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-d-oliose (sugar A) and 4-O-methyl-d-oliose (sugar B), and a trisaccharide of d-olivose (sugar C), d-olivose (sugar D), and 4-O-acetyl-l-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two d-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 biosynthesis.
Lutz Heide - One of the best experts on this subject based on the ideXlab platform.
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improved mutasynthetic approaches for the production of modified aminocoumarin antibiotics
Chemistry & Biology, 2007Co-Authors: Christine Anderle, Susanne Hennig, Bertolt Gust, Bernd Kammerer, Shuming Li, Ludger A. Wessjohann, Lutz HeideAbstract:Summary This study reports improved mutasynthetic approaches for the production of aminocoumarin antibiotics. Previously, the mutasynthetic production of aminocoumarins with differently substituted benzoyl moieties was limited by the substrate specificity of the amide synthetase CloL. We expressed two amide synthetases with different substrate specificity, CouL and SimL, in appropriately engineered producer strains. After feeding of precursor analogs that were not accepted by CloL, but by SimL or CouL, a range of aminocoumarins, unattainable in our previous experiments, was produced and isolated in preparative amounts. Further, we developed a two-stage mutasynthesis procedure for the production of hybrid antibiotics that showed the substitution pattern of novobiocin in the aminocoumarin moiety and that of clorobiocin in the Deoxysugar moiety. The substitution pattern of the benzoyl moiety was determined by external addition of an appropriate precursor. Twenty-five aminocoumarin compounds were prepared by these methods, and their structures were elucidated with mass and 1 H-NMR spectroscopy.
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Biosynthesis of clorobiocin: investigation of the transfer and methylation of the pyrrolyl-2-carboxyl moiety
Archives of Microbiology, 2007Co-Authors: Christine Anderle, Bertolt Gust, Bernd Kammerer, Tanja Gulder, Gerhard Bringmann, Lutz HeideAbstract:Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a Deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the Deoxysugar. In this study, pyrrolyl-2-carboxyl- N -acetylcysteamine thioester was synthesized and fed to cloN1 ^ − , cloN2 ^ − and cloN7 ^ − mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1 ^ − and cloN2 ^ − mutants, but not in the cloN7 ^ − mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6 , suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.
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biosynthesis of the unusual 5 5 gem dimethyl Deoxysugar noviose investigation of the c methyltransferase gene clou
Microbiology, 2006Co-Authors: Anja Freitag, Shuming Li, Lutz HeideAbstract:The aminocoumarin antibiotic clorobiocin contains an unusual branched Deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this Deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the Deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1H-NMR and mass spectroscopy and their antibacterial activity was assayed.
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acyl transfer in clorobiocin biosynthesis involvement of several proteins in the transfer of the pyrrole 2 carboxyl moiety to the Deoxysugar
ChemBioChem, 2005Co-Authors: Anja Freitag, Shuming Li, Emmanuel Wemakor, Lutz HeideAbstract:: Clorobiocin is an aminocoumarin antibiotic containing a pyrrole-2-carboxyl moiety, attached through an ester bond to a Deoxysugar. The pyrrole moiety is important for the binding of the antibiotic to its biological target, gyrase. The complete biosynthetic gene cluster for clorobiocin has been cloned and sequenced from the natural producer, Streptomyces roseochromogenes DS 12.976. In this study, the genes cloN1 and cloN7 were deleted separately from a cosmid containing the complete clorobiocin cluster. The modified cosmids were introduced into the genome of the heterologous host Streptomyces coelicolor M512 by using the integration functions of the PhiC31 phage. While a heterologous producer strain harbouring the intact clorobiocin biosynthetic gene cluster accumulated clorobiocin, the cloN1- and cloN7-defective integration mutants accumulated a clorobiocin derivative that lacked the pyrrole-2-carboxyl moiety, while also producing free pyrrole-2-carboxylic acid. The structures of these metabolites were confirmed by NMR and MS analysis. These results showed that CloN1 and CloN7, together with the previously investigated CloN2, are involved in the transfer of the pyrrole-2-carboxyl moiety to the Deoxysugar of clorobiocin. A possible mechanism for the role of these three proteins in the acyl-transfer process is suggested.
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Metabolic Engineering of Aminocoumarins: Inactivation of the Methyltransferase Gene cloP and Generation of New Clorobiocin Derivatives in a Heterologous Host
ChemBioChem, 2005Co-Authors: Anja Freitag, Heike Rapp, Lutz Heide, Shuming LiAbstract:Aminocoumarin antibiotics are highly potent inhibitors of bacterial gyrase and represent a class of antibiotics that are very suitable for the generation of new compounds by metabolic engineering. In this study, the putative methyltransferase gene cloP in the biosynthetic gene cluster of clorobiocin was inactivated. Expression of the modified gene cluster in the heterologous host Streptomyces coelicolor M512 gave three new aminocoumarin antibiotics. The structures of the new compounds were elucidated by MS and 1H NMR, and their antibacterial activities were determined. All three compounds lacked clorobiocin's methyl group at 4-OH of the Deoxysugar moiety, noviose. They differed from each other in the position of the 5-methylpyrrole-2-carbonyl group, which was found to be attached to either 2-OH, 3-OH or 4-OH of noviose. Attachment at 4-OH resulted in the highest antibacterial activity. This is the first time that an aminocoumarin antibiotic acylated at 4-OH in noviose has been detected.
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, Bernd Kammerer, Jae Kyung Sohng, José A. Salas, Emmanuel Wemakor, Stefanie Siebenberg, 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, Alfredo F Brana, 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: Maria S Perez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alfredo F Brana, 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, Alfredo F Brana, 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.
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, Madan K Kharel, Pallab Pahari, 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: Maria S Perez, Felipe Lombó, José A. Salas, Jurgen Rohr, Alfredo F Brana, 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, Alfredo F Brana, 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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Deoxysugar transfer during chromomycin a3 biosynthesis in streptomyces griseus subsp griseus new derivatives with antitumor activity
Applied and Environmental Microbiology, 2006Co-Authors: Nuria Menendez, José A. Salas, Jurgen Rohr, Alfredo F Brana, Mohammad Nurealam, Carsten Fischer, Carmen MéndezAbstract:Chromomycin A3 is an antitumor drug produced by Streptomyces griseus subsp. griseus. It consists of a tricyclic aglycone with two aliphatic side chains and two O-glycosidically linked saccharide chains, a disaccharide of 4-O-acetyl-d-oliose (sugar A) and 4-O-methyl-d-oliose (sugar B), and a trisaccharide of d-olivose (sugar C), d-olivose (sugar D), and 4-O-acetyl-l-chromose B (sugar E). The chromomycin gene cluster contains four glycosyltransferase genes (cmmGI, cmmGII, cmmGIII, and cmmGIV), which were independently inactivated through gene replacement, generating mutants C60GI, C10GII, C10GIII, and C10GIV. Mutants C10GIV and C10GIII produced the known compounds premithramycinone and premithramycin A1, respectively, indicating the involvement of CmmGIV and CmmGIII in the sequential transfer of sugars C and D and possibly also of sugar E of the trisaccharide chain, to the 12a position of the tetracyclic intermediate premithramycinone. Mutant C10GII produced two new tetracyclic compounds lacking the disaccharide chain at the 8 position, named prechromomycin A3 and prechromomycin A2. All three compounds accumulated by mutant C60GI were tricyclic and lacked sugar B of the disaccharide chain, and they were named prechromomycin A4, 4A-O-deacetyl-3A-O-acetyl-prechromomycin A4, and 3A-O-acetyl-prechromomycin A4. CmmGII and CmmGI are therefore responsible for the formation of the disaccharide chain by incorporating, in a sequential manner, two d-oliosyl residues to the 8 position of the biosynthetic intermediate prechromomycin A3. A biosynthetic pathway is proposed for the glycosylation events in chromomycin A3 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, Alfredo F Brana, 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.