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José A. Salas - One of the best experts on this subject based on the ideXlab platform.
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Oviedomycin, an unusual angucyclinone encoded by genes of the Oleandomycin-producer Streptomyces antibioticus ATCC11891.
Journal of natural products, 2002Co-Authors: Carmen Méndez, Alfredo F. Braña, José A. Salas, Eva Künzel, Fredilyn Lipata, Felipe Lombó, William E. Cotham, Michael D. Walla, Daniel W. Bearden, Jürgen RohrAbstract:Our investigations on the discovery of novel natural metabolites using type II polyketide synthase gene probes (actI/III) yielded an unusual angucyclinone, oviedomycin (2), when applied to the Oleandomycin (1) producer Streptomyces antibioticus ATCC11891. The novel natural product was produced using S. albus R(-)M(-) as a host strain, into which a cosmid containing the oviedomycin gene cluster was transformed. Its structure was elucidated by NMR spectroscopy and mass spectrometry.
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JMMB Communication
1999Co-Authors: Macrolide Biosynthetic Pathways, Carmen Méndez, Alfredo F. Braña, Jürgen Rohr, Ignacio Aguirrezabalaga, Leticia Rodriguez, Carsten Oelkers, José A. SalasAbstract:Elloramycin and Oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and Oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic Oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandros
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two glycosyltransferases and a glycosidase are involved in Oleandomycin modification during its biosynthesis by streptomyces antibioticus
Molecular Microbiology, 1998Co-Authors: Luis M. Quirós, Carmen Méndez, Carlos Olano, Ignacio Aguirrezabalaga, José A. SalasAbstract:A 5.2 kb region from the Oleandomycin gene cluster in Streptomyces antibioticus located between the Oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI, oleN2 and oleR). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus. The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI, oleR and oleD genes were expressed in Streptomyces lividans. OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed Oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for Oleandomycin. OleR showed glycosidase activity converting glycosylated Oleandomycin into active Oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of Oleandomycin.
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Analysis of a Streptomyces antibioticus chromosomal region involved in Oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring.
Molecular & general genetics : MGG, 1998Co-Authors: Carlos Olano, Carmen Méndez, Ana Rodríguez, J.-m. Michel, M.-c. Raynal, José A. SalasAbstract:A 6-kb region from the chromosome of Streptomyces antibioticus, an Oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the Oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an Oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the Oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an Oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the Oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.
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characterization of the atpase activity of the n terminal nucleotide binding domain of an abc transporter involved in Oleandomycin secretion by streptomyces antibioticus
Fems Microbiology Letters, 1996Co-Authors: Gerardo Aparicio, Carmen Méndez, Andre Buche, José A. SalasAbstract:The oleB gene of Streptomyces antibioticus, Oleandomycin producer, encodes an ABC transporter containing two putative ATP-binding domains and is involved in Oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N-terminal nucleotide-binding domain of OleB (OleB′) as a fusion protein to a maltose-binding protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg2+ ions. ATPase activity was resistant to specific inhibitors of P-, F-, and V-type ATPase whereas sodium azide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB′ protein, to Gin or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP-hydrolysing enzymes, including ion-translocating ATPases or ABC-traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.
Carmen Méndez - One of the best experts on this subject based on the ideXlab platform.
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Oviedomycin, an unusual angucyclinone encoded by genes of the Oleandomycin-producer Streptomyces antibioticus ATCC11891.
Journal of natural products, 2002Co-Authors: Carmen Méndez, Alfredo F. Braña, José A. Salas, Eva Künzel, Fredilyn Lipata, Felipe Lombó, William E. Cotham, Michael D. Walla, Daniel W. Bearden, Jürgen RohrAbstract:Our investigations on the discovery of novel natural metabolites using type II polyketide synthase gene probes (actI/III) yielded an unusual angucyclinone, oviedomycin (2), when applied to the Oleandomycin (1) producer Streptomyces antibioticus ATCC11891. The novel natural product was produced using S. albus R(-)M(-) as a host strain, into which a cosmid containing the oviedomycin gene cluster was transformed. Its structure was elucidated by NMR spectroscopy and mass spectrometry.
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JMMB Communication
1999Co-Authors: Macrolide Biosynthetic Pathways, Carmen Méndez, Alfredo F. Braña, Jürgen Rohr, Ignacio Aguirrezabalaga, Leticia Rodriguez, Carsten Oelkers, José A. SalasAbstract:Elloramycin and Oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and Oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic Oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandros
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two glycosyltransferases and a glycosidase are involved in Oleandomycin modification during its biosynthesis by streptomyces antibioticus
Molecular Microbiology, 1998Co-Authors: Luis M. Quirós, Carmen Méndez, Carlos Olano, Ignacio Aguirrezabalaga, José A. SalasAbstract:A 5.2 kb region from the Oleandomycin gene cluster in Streptomyces antibioticus located between the Oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI, oleN2 and oleR). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus. The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI, oleR and oleD genes were expressed in Streptomyces lividans. OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed Oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for Oleandomycin. OleR showed glycosidase activity converting glycosylated Oleandomycin into active Oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of Oleandomycin.
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Analysis of a Streptomyces antibioticus chromosomal region involved in Oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring.
Molecular & general genetics : MGG, 1998Co-Authors: Carlos Olano, Carmen Méndez, Ana Rodríguez, J.-m. Michel, M.-c. Raynal, José A. SalasAbstract:A 6-kb region from the chromosome of Streptomyces antibioticus, an Oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the Oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an Oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the Oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an Oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the Oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.
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characterization of the atpase activity of the n terminal nucleotide binding domain of an abc transporter involved in Oleandomycin secretion by streptomyces antibioticus
Fems Microbiology Letters, 1996Co-Authors: Gerardo Aparicio, Carmen Méndez, Andre Buche, José A. SalasAbstract:The oleB gene of Streptomyces antibioticus, Oleandomycin producer, encodes an ABC transporter containing two putative ATP-binding domains and is involved in Oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N-terminal nucleotide-binding domain of OleB (OleB′) as a fusion protein to a maltose-binding protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg2+ ions. ATPase activity was resistant to specific inhibitors of P-, F-, and V-type ATPase whereas sodium azide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB′ protein, to Gin or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP-hydrolysing enzymes, including ion-translocating ATPases or ABC-traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.
Carlos Olano - One of the best experts on this subject based on the ideXlab platform.
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two glycosyltransferases and a glycosidase are involved in Oleandomycin modification during its biosynthesis by streptomyces antibioticus
Molecular Microbiology, 1998Co-Authors: Luis M. Quirós, Carmen Méndez, Carlos Olano, Ignacio Aguirrezabalaga, José A. SalasAbstract:A 5.2 kb region from the Oleandomycin gene cluster in Streptomyces antibioticus located between the Oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI, oleN2 and oleR). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus. The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI, oleR and oleD genes were expressed in Streptomyces lividans. OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed Oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for Oleandomycin. OleR showed glycosidase activity converting glycosylated Oleandomycin into active Oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of Oleandomycin.
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Analysis of a Streptomyces antibioticus chromosomal region involved in Oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring.
Molecular & general genetics : MGG, 1998Co-Authors: Carlos Olano, Carmen Méndez, Ana Rodríguez, J.-m. Michel, M.-c. Raynal, José A. SalasAbstract:A 6-kb region from the chromosome of Streptomyces antibioticus, an Oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the Oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an Oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the Oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an Oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the Oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.
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a second abc transporter is involved in Oleandomycin resistance and its secretion by streptomyces antibioticus
Molecular Microbiology, 1995Co-Authors: Carlos Olano, Carmen Méndez, Ana Rodríguez, José A. SalasAbstract:Summary A 3.2 kb Sstl-Sphl DNA fragment of Streptomyces antibioticus, an Oleandomycin producer, conferring resistance to Oleandomycin was sequenced and found to contain an open reading frame of 1710 bp (oleB). Its deduced gene product (OleB) showed a high degree of similarity with other proteins belonging to the ABC-transporter superfamily including the gene product of another Oleandomycin-resistance gene (OleC). The OleB protein contains two ATP-binding domains, each of approximately 200 amino acids in length, and no hydrophobic transmembrane regions. Functional analysis of the oleB gene was carried out by deleting specific regions of the gene and assaying for Oleandomycin resistance. These experiments showed that either the first or the second half of the gene containing only one ATP-binding domain was sufficient to confer resistance to Oleandomycin. The gene oleB was expressed in Escherichia coli fused to a maltose-binding protein (MBP) using the pMal-c2 vector. The MBP-OleB hybrid protein was purified by affinity chromatography on an amylose resin and polyclonal antibodies were raised against the fusion protein. These were used to monitor the biosynthesis and physical location of OleB during growth. By Western analysis, the OleB protein was detected both in the soluble and in the membrane fraction and its synthesis paralleled Oleandomycin biosynthesis. It was also shown that a Streptomyces albus strain, containing both a glycosyltransferase (OleD) able to inactivate Oleandomycin and the OleB protein, was capable of glycosylating Oleandomycin and secreting the inactive glycosylated molecule. It is proposed that OleB constitutes the secretion system by which Oleandomycin or its inactive glycosylated form could be secreted by S. antibioticus.
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A cytochrome P450-like gene possibly involved in Oleandomycin biosynthesis by Streptomyces antibioticus
FEMS microbiology letters, 1995Co-Authors: Ana Rodríguez, Carmen Méndez, Carlos Olano, C. Richard Hutchinson, J.a. SalasAbstract:A cosmid clone from an Oleandomycin producer, Streptomyces antibioticus, contains a large open reading frame encoding a type I polyketide synthase subunit and an Oleandomycin resistance gene (oleB). Sequencing of a 1.4-kb DNA fragment adjacent to oleB revealed the existence of an open reading frame (oleP) encoding a protein similar to several cytochrome P450 monooxygenases from different sources, including the products of the eryF and eryK genes from Saccharopolyspora erythraea that participate in erythromycin biosynthesis. The oleP gene was expressed in Escherichia coli as a fusion protein to a maltose-binding protein. Using polyclonal antibodies against this fusion protein it was observed that the synthesis of the cytochrome P450 was in parallel to that of Oleandomycin. The cytochrome P450 encoded by the oleP gene could be responsible for the epoxidation of carbon 8 of the Oleandomycin lactone ring.
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Research letterA cytochrome P450-like gene possibly involved in Oleandomycin biosynthesis by Streptomyces antibioticus
Fems Microbiology Letters, 1995Co-Authors: Ana Rodríguez, Carmen Méndez, Carlos Olano, C. Richard Hutchinson, J.a. SalasAbstract:A cosmid clone from an Oleandomycin producer, Streptomyces antibioticus, contains a large open reading frame encoding a type I polyketide synthase subunit and an Oleandomycin resistance gene (oleB). Sequencing of a 1.4-kb DNA fragment adjacent to oleB revealed the existence of an open reading frame (oleP) encoding a protein similar to several cytochrome P450 monooxygenases from different sources, including the products of the eryF and eryK genes from Saccharopolyspora erythraea that participate in erythromycin biosynthesis. The oleP gene was expressed in Escherichia coli as a fusion protein to a maltose-binding protein. Using polyclonal antibodies against this fusion protein it was observed that the synthesis of the cytochrome P450 was in parallel to that of Oleandomycin. The cytochrome P450 encoded by the oleP gene could be responsible for the epoxidation of carbon 8 of the Oleandomycin lactone ring.
Predrag Novak - One of the best experts on this subject based on the ideXlab platform.
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Epitope Mapping of Macrolide Antibiotics to Bovine Serum Albumin by Saturation Transfer Difference NMR Spectroscopy
Croatica Chemica Acta, 2007Co-Authors: Predrag Novak, Predrag Tepeš, Vedrana LazićAbstract:Saturation transfer difference NMR spectroscopy was employed to characterize epitopes of macrolide antibiotics, azithromycin, Oleandomycin and telithromycin binding to bovine serum albumin. The structural parts of azithromycin and Oleandomycin in intimate contact with bovine serum albumin were found to be similar while those of telithromycin showed similarities but also some differences. The latter were mostly due to different structural elements of antibiotics that interact with the protein, especially the alkyl-heteroaryl side chain in telithromycin and cladinose and desosamine sugars in azithromycin and Oleandomycin. The epitope maps as determined in this study can contribute to better understanding of the overall bioactivity of macrolides.
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Epitope Mapping of Macrolide Antibiotics to Bovine Serum Albumin by Saturation Transfer Difference NMR Spectroscopy*
2007Co-Authors: Predrag Novak, Predrag TepeAbstract:Saturation transfer difference NMR spectroscopy was employed to characterize epitopes of macrolide antibiotics, azithromycin, Oleandomycin and telithromycin binding to bovine serum albumin. The structural parts of azithromycin and Oleandomycin in intimate contact with bo-vine serum albumin were found to be similar while those of telithromycin showed similarities but also some differences. The latter were mostly due to different structural elements of antibi-otics that interact with the protein, especially the alkyl-heteroaryl side chain in telithromycin and cladinose and desosamine sugars in azithromycin and Oleandomycin. The epitope maps as determined in this study can contribute to better understanding of the overall bioactivity of macrolides. Keywords: STD NMR spectroscopy macrolide antibiotics albumin epitope mapping * Dedicated to Professor Haruo Hosoya in happy celebration of his 70th birthday
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Systematic approach to understanding macrolide-ribosome interactions: NMR and modeling studies of Oleandomycin and its derivatives
The journal of physical chemistry. A, 2006Co-Authors: Predrag Novak, Predrag Tepeš, Iva Tatić, Sanja Koštrun, Jill BarberAbstract:The three-dimensional structures of Oleandomycin (1) and its derivatives Oleandomycin-9-oxime (2) and 10,11-anhydroOleandomycin (3) were determined in different solvents by the combined use of NMR and molecular modeling methods. The experimental NMR data were compared with the results of molecular modeling and known crystal structures of the related molecules. It was shown that the dominant conformation of the lactone ring is the folded-out conformation with some amounts of the folded-in one depending on the solvent and temperature, while desosamine and cladinose sugars adopt the usual chair conformations. Modeling calculations provided evidence for conformational changes in the upper lactone region as well. Saturation transfer difference (STD) NMR experiments have provided information on the binding epitopes of 1-3 in complexes with E. coli ribosomes. The obtained molecular surfaces in close contact with ribosomes were compared with recently available 3D structures of the related macrolide-ribosome complexes, and the observed differences were discussed. The knowledge gained from this study can serve as a platform for the design of novel macrolides with an improved biological profile.
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A Systematic Approach to Understanding Ribosome-Macrolide Interactions: NMR and Modeling Studies of Oleandomycin and Its Derivatives
Journal of Physical Chemistry A, 2006Co-Authors: Predrag Novak, Predrag Tepeš, Iva Tatić, Sanja Koštrun, Jill BarberAbstract:The three-dimensional structures of Oleandomycin (1) and its derivatives Oleandomycin-9-oxime (2) and 10, 11- anhydroOleandomycin (3) were determined in different solvents by the combined use of NMR and molecular modeling methods. The experimental NMR data were compared with the results of molecular modeling and known crystal structures of the related molecules. It was shown that the dominant conformation of the lactone ring is the folded-out conformation with some amounts of the folded-in one depending on the solvent and temperature, while desosamine and cladinose sugars adopt the usual chair conformations. Modeling calculations provided evidence for conformational changes in the upper lactone region as well. Saturation transfer difference (STD) NMR experiments have provided information on the binding epitopes of 1-3 in complexes with E. coli ribosomes. The obtained molecular surfaces in close contact with ribosomes were compared with recently available 3D structures of the related macrolide-ribosome complexes, and the observed differences were discussed. The knowledge gained from this study can serve as a platform for the design of novel macrolides with an improved
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Conformational analysis of Oleandomycin and its 8-methylene-9-oxime derivative by NMR and molecular modelling.
Organic & biomolecular chemistry, 2004Co-Authors: Predrag Novak, Predrag Tepeš, Zrinka Banić Tomišić, Gorjana Lazarevski, Janez Plavec, Gordana TurkaljAbstract:Conformations of the 14-membered macrolide antibiotic Oleandomycin and its 8-methylene-9-oxime derivative were determined in various solvents. The experimental NMR data—coupling constants and NOE contacts—were compared with the results of molecular modelling—molecular mechanics calculations and molecular dynamics simulations. The conformational changes, on the right-hand side of the 14-membered ring, affected mostly the 3JH2,H3 values and NOE crosspeaks H3 or H4 to H11. Oleandomycin was found to be present predominantly in the C3–C5 folded-in conformations in DMSO-d6 solution, whereas in buffered D2O, acetone-d6 and CDCl3, there was a mixture of folded-in and folded-out conformational families. The predominant conformation of the 8-methylene-Oleandomycin-9-oxime derivative in solution was a folded-out one although different amounts of folded-in conformation were also present depending on the solvent. Oleandrose and desosamine sugar moieties adopted the usual and expected chair conformation. The conformation around the glycosidic bonds, governing the relative orientation of sugars vs. the lactone ring, showed a certain flexibility within two conformationally close families. We believe that by combining the experimental NMR data and the molecular modelling techniques, as reported in this paper, we have made significant progress in understanding the conformational behaviour and properties of macrolides. Our belief is based on our own current studies on Oleandomycins as well as on the previously reported results and best practices concerning other macrolides. A rational for macrolide conformational studies and advances in methodology has been suggested accordingly.
Carmen Vilches - One of the best experts on this subject based on the ideXlab platform.
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characterisation of a streptomyces antibioticus gene encoding a type i polyketide synthase which has an unusual coding sequence
Molecular Genetics and Genomics, 1994Co-Authors: David G Swan, Carmen Méndez, Carmen Vilches, Ana Rodríguez, José A. SalasAbstract:A gene (ORFB) from Streptomyces antibioticus (an Oleandomycin producer) encoding a large, multifunctional polyketide synthase (PKS) was cloned and sequenced. Its product shows an internal duplication and a close similarity to the third subunit of the PKS involved in erythromycin biosynthesis by Saccharopolyspora erythraea, showing the equivalent nine active site domains in the same order along the polypeptide. An unusual feature of this ORF is the GC content of most of the sequence, which is surprisingly low, for a Streptomyces gene; the large number of codons with T in the third position is particularly striking. The last 800 by of the gene stand out as being normal in their GC content, this region corresponding almost exactly to the thioesterase domain of the gene and suggesting that this domain was a late addition to the PKS. Based on the high degree of similarity between the ORFB product and the third subunit of the erythromycin PKS and the occurrence nearby of a gene conferring Oleandomycin resistance, it is possible that this gene might be involved in the biosynthesis of the Oleandomycin lactone ring.
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Intracellular glycosylation and active efflux as mechanisms for resistance to Oleandomycin in Streptomyces antibioticus, the producer organism.
Microbiologia (Madrid Spain), 1994Co-Authors: José A. Salas, Carmen Méndez, Ana Rodríguez, Carlos Olano, César Hernández, Luis M. Quirós, Carmen VilchesAbstract:Abstract Resistance to macrolides in producing organisms can be achieved by target site modification, intracellular inactivation of the antibiotic or active efflux mechanisms for the excretion of the antibiotic. The Oleandomycin producer, Streptomyces antibioticus, possesses Oleandomycin-sensitive ribosomes all along the cell cycle. However, it contains an intracellular glycosyltransferase capable of inactivating Oleandomycin in the presence of UDP-glucose as cofactor. The correspondent gene (oleD) has been cloned and sequenced and the glycosyltransferase purified. Two other genes (oleB and oleC) that confer Oleandomycin resistance have been cloned and characterized and both encode ABC (ATP-Binding Cassette) transporters. These may constitute the excretion mechanism throughout which the glycosylated Oleandomycin is excreted. A second enzyme activity has been purified from culture supernatants of the Oleandomycin producer that releases the glucose from the inactive glycosylated Oleandomycin generating active antibiotic. This enzyme would probably catalyse the last step in the biosynthesis of Oleandomycin.
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streptomyces antibioticus contains at least three Oleandomycin resistance determinants one of which shows similarity with proteins of the abc transporter superfamily
Molecular Microbiology, 1993Co-Authors: Ana Ma Rodriguez, Carmen Méndez, Carmen Vilches, Carlos Olano, José A. SalasAbstract:Summary Three different DNA fragments of an Oleandomycin producer, Streptomyces antibioticus, conferring Oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These Oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans-membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1-Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to Oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.
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Streptomyces antibioticus contains at least three Oleandomycin‐resistance determinants, one of which shows similarity with proteins of the ABC‐transporter superfamily
Molecular microbiology, 1993Co-Authors: Ana Ma Rodriguez, Carmen Méndez, Carmen Vilches, Carlos Olano, José A. SalasAbstract:Summary Three different DNA fragments of an Oleandomycin producer, Streptomyces antibioticus, conferring Oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These Oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans-membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1-Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to Oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.
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Role of glycosylation and deglycosylation in biosynthesis of and resistance to Oleandomycin in the producer organism, Streptomyces antibioticus.
Journal of bacteriology, 1992Co-Authors: Carmen Vilches, Carmen Méndez, César Hernández, José A. SalasAbstract:Cell extracts of Streptomyces antibioticus, an Oleandomycin producer, can inactivate Oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 29-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated Oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the Oleandomycin biosynthetic pathway. Images
