| 1. |
Blanco G,
Parra F,
Méndez C,
Salas JA,
( 1992 ) The nucleotide sequence of the L10 equivalent ribosomal protein gene of Streptomyces antibioticus. PMID : 1408837 : DOI : 10.1093/nar/20.19.5223 PMC : PMC334308 Abstract >>
N/A
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2. |
Funa N,
Ohnishi Y,
Ebizuka Y,
Horinouchi S,
( 2002 ) Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. PMID : 12139488 : DOI : 10.1042/BJ20020953 PMC : PMC1222926 Abstract >>
RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.
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3. |
Galm U,
Schimana J,
Fiedler HP,
Schmidt J,
Li SM,
Heide L,
( 2002 ) Cloning and analysis of the simocyclinone biosynthetic gene cluster of Streptomyces antibioticus T? 6040. PMID : 12115055 : DOI : 10.1007/s00203-002-0429-z Abstract >>
The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusT? 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.
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4. |
Trefzer A,
Pelzer S,
Schimana J,
Stockert S,
Bihlmaier C,
Fiedler HP,
Welzel K,
Vente A,
Bechthold A,
( 2002 ) Biosynthetic gene cluster of simocyclinone, a natural multihybrid antibiotic. PMID : 11959542 : DOI : 10.1128/aac.46.5.1174-1182.2002 PMC : PMC127163 Abstract >>
The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus T?6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.
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5. |
Colombo V,
Fernández-de-Heredia M,
Malpartida F,
( 2001 ) A polyketide biosynthetic gene cluster from Streptomyces antibioticus includes a LysR-type transcriptional regulator. PMID : 11700358 : DOI : 10.1099/00221287-147-11-3083 Abstract >>
In the search for Type II polyketide synthases (PKSs) a DNA fragment was isolated from Streptomyces antibioticus ATCC 11891 (a producer of oleandomycin). DNA sequencing of the cloned fragment revealed six complete ORFs whose deduced products showed similarities to those of other genes known to be involved in polyketide biosynthesis. Several S. coelicolor strains mutated in different steps of actinorhodin biosynthesis (actI, actIII, actV(A) and actVII) were complemented by the cloned genes, suggesting that the isolated genes encode an aromatic polyketide of unknown structure and function. The cluster also contains a putative LysR-type transcriptional regulator (ORF0), which controls PKS gene expression in a heterologous host. DNA binding assays and transcriptional analysis suggest that the pathway-specific regulator for actinorhodin biosynthesis (actII-ORF4) is also involved in the expression of the cloned PKS in the host strain.
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6. |
Rodríguez L,
Rodríguez D,
Olano C,
Braña AF,
Méndez C,
Salas JA,
( 2001 ) Functional analysis of OleY L-oleandrosyl 3-O-methyltransferase of the oleandomycin biosynthetic pathway in Streptomyces antibioticus. PMID : 11514520 : DOI : 10.1128/jb.183.18.5358-5363.2001 PMC : PMC95419 Abstract >>
Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl- and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co(2+). The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.
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7. |
Bralley P,
Jones GH,
( 2001 ) Transcriptional analysis and regulation of the sigma-E gene of Streptomyces antibioticus. PMID : 11342219 : DOI : 10.1016/s0167-4781(00)00257-8 Abstract >>
We report here the mapping of the transcriptional start point and identification of the promoter for the sigE gene of Streptomyces antibioticus. Sequence analysis revealed a conserved genetic organization of five genes encompassing sigE in S. antibioticus and S. coelicolor. Upstream of sigE a number of direct repeats, while conserved in both species, are arranged differently. Gel shift analysis demonstrated binding of a component of both S. antibioticus and S. coelicolor crude protein extracts to a 30 bp sequence encompassing one repeat, the A-rich box. Deletion analysis in promoter probes showed that maximal activity of the S. antibioticus promoter depends upon the presence of the sequence surrounding the A-rich box, as well as the region further upstream carrying other direct repeats.
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8. |
Symmons MF,
Jones GH,
Luisi BF,
( 2000 ) A duplicated fold is the structural basis for polynucleotide phosphorylase catalytic activity, processivity, and regulation. PMID : 11080643 : Abstract >>
Polynucleotide phosphorylase (PNPase) is a polyribonucleotide nucleotidyl transferase (E.C.2.7.7.8) that degrades mRNA in prokaryotes. Streptomyces antibioticus PNPase also assays as a guanosine 3'-diphosphate 5'-triphosphate (pppGpp) synthetase (E.C.2.7.6.5). It may function to coordinate changes in mRNA lifetimes with pppGpp levels during the Streptomyces lifecycle. The structure of S. antibioticus PNPase without bound RNA but with the phosphate analog tungstate bound at the PNPase catalytic sites was determined by X-ray crystallography and shows a trimeric multidomain protein with a central channel. The structural core has a novel duplicated architecture formed by association of two homologous domains. The tungstate derivative structure reveals the PNPase active site in the second of these core domains. Structure-based sequence analysis suggests that the pppGpp synthetase active site is located in the first core domain. This is the first structure of a PNPase and shows the structural basis for the trimer assembly, the arrangement of accessory RNA binding domains, and the likely catalytic residues of the PNPase active site. A possible function of the trimer channel is as a contribution to both the processivity of degradation and the regulation of PNPase action by RNA structural elements.
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9. |
Jones GH,
( 1999 ) relA is required for actinomycin production in Streptomyces antibioticus. PMID : 10368159 : PMC : PMC93862 Abstract >>
The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an Mr of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relA produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.
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10. |
Huergo J,
Connolly BA,
Sanchez J,
( 1999 ) Purification, characterization, and role of nucleases and serine proteases in Streptomyces differentiation. Analogies with the biochemical processes described in late steps of eukaryotic apoptosis. PMID : 10400660 : DOI : 10.1074/jbc.274.29.20366 Abstract >>
Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.
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11. |
Luzhetskyy A,
Weiss H,
Charge A,
Welle E,
Linnenbrink A,
Vente A,
Bechthold A,
( 2007 ) A strategy for cloning glycosyltransferase genes involved in natural product biosynthesis. PMID : 17497146 : DOI : 10.1007/s00253-007-0950-8 Abstract >>
The soil-borne and marine gram-positive Actinomycetes are a particularly rich source of carbohydrate-containing metabolites. With the advent of molecular tools and recombinant methods applicable to Actinomycetes, it has become feasible to investigate the biosynthesis of glycosylated compounds at genetic and biochemical levels, which has finally set the basis for engineering novel natural product derivatives. Glycosyltransferases (GT) are key enzymes for the biosynthesis of many valuable natural products that contain sugar moieties and they are most important for drug engineering. So far, the direct cloning of unknown glycosyltransferase genes by polymerase chain reaction (PCR) has not been described because glycosyltransferases do not share strongly conserved amino acid regions. In this study, we report a method for cloning of novel so far unidentified glycosyltransferase genes from different Actinomycetes strain. This was achieved by designing primers after a strategy named consensus-degenerate hybrid oligonucleotide primer (CODEHOP). Using this approach, 22 novel glycosyltransferase encoding genes putatively involved in the decoration of polyketides were cloned from the genomes of 10 Actinomycetes. In addition, a phylogenetic analysis of glycosyltransferases from Actinomycetes is shown in this paper.
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12. |
Bolam DN,
Roberts S,
Proctor MR,
Turkenburg JP,
Dodson EJ,
Martinez-Fleites C,
Yang M,
Davis BG,
Davies GJ,
Gilbert HJ,
( 2007 ) The crystal structure of two macrolide glycosyltransferases provides a blueprint for host cell antibiotic immunity. PMID : 17376874 : DOI : 10.1073/pnas.0607897104 PMC : PMC1838483 DOI : 10.1073/pnas.0607897104 PMC : PMC1838483 Abstract >>
Glycosylation of macrolide antibiotics confers host cell immunity from endogenous and exogenous agents. The Streptomyces antibioticus glycosyltransferases, OleI and OleD, glycosylate and inactivate oleandomycin and diverse macrolides including erythromycin, respectively. The structure of these enzyme-ligand complexes, in tandem with kinetic analysis of site-directed variants, provide insight into the interaction of macrolides with their synthetic apparatus. Erythromycin binds to OleD and the 23S RNA of its target ribosome in the same conformation and, although the antibiotic contains a large number of polar groups, its interaction with these macromolecules is primarily through hydrophobic contacts. Erythromycin and oleandomycin, when bound to OleD and OleI, respectively, adopt different conformations, reflecting a subtle effect on sugar positioning by virtue of a single change in the macrolide backbone. The data reported here provide structural insight into the mechanism of resistance to both endogenous and exogenous antibiotics, and will provide a platform for the future redesign of these catalysts for antibiotic remodelling.
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13. |
Manteca A,
Pelaez AI,
Zardoya R,
Sanchez J,
( 2006 ) Actinobacteria cyclophilins: phylogenetic relationships and description of new class- and order-specific paralogues. PMID : 17103061 : DOI : 10.1007/s00239-005-0130-3 Abstract >>
Cyclophilins are folding helper enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins. They are ubiquitous proteins present in almost all living organisms analyzed to date, with extremely rare exceptions. Few cyclophilins have been described in Actinobacteria, except for three reported in the genus Streptomyces and another one in Mycobacterium tuberculosis. In this study, we performed a complete phylogenetic analysis of all Actinobacteria cyclophilins available in sequence databases and new Streptomyces cyclophilin genes sequenced in our laboratory. Phylogenetic analyses of cyclophilins recovered six highly supported groups of paralogy. Streptomyces appears as the bacteria having the highest cyclophilin diversity, harboring proteins from four groups. The first group was named "A" and is made up of highly conserved cytosolic proteins of approximately 18 kDa present in all Actinobacteria. The second group, "B," includes cytosolic proteins widely distributed throughout the genus Streptomyces and closely related to eukaryotic cyclophilins. The third group, "M" cyclophilins, consists of high molecular mass cyclophilins (approximately 30 kDa) that contain putative membrane binding domains and would constitute the only membrane cyclophilins described to date in bacteria. The fourth group, named "C" cyclophilins, is made up of proteins of approximately 18 kDa that are orthologous to Gram-negative proteobacteria cyclophilins. Ancestral character reconstruction under parsimony was used to identify shared-derived (and likely functionally important) amino acid residues of each paralogue. Southern and Western blot experiments were performed to determine the taxonomic distribution of the different cyclophilins in Actinobacteria.
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14. |
Jia XY,
Tian ZH,
Shao L,
Qu XD,
Zhao QF,
Tang J,
Tang GL,
Liu W,
( 2006 ) Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis. PMID : 16793515 : DOI : 10.1016/j.chembiol.2006.03.008 Abstract >>
The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM 40725, and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation. Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames, 35 of which were defined to constitute the CHL gene cluster. An assembly model for CHL biosynthesis from D-olivose, 2-methoxy-5-chloro-6-methylsalicyclic acid, and chlorothricolide building blocks was proposed. This work represents cloning of a gene cluster for spirotetronate antibiotic biosynthesis and sets the stage to investigate the unusual macrolide biosynthesis including tandem Diels-Alder cyclizations, Baeyer-Villiger oxidation, and incorporation of an enoylpyruvate unit.
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15. |
Smith AW,
Camara-Artigas A,
Wang M,
Allen JP,
Francisco WA,
( 2006 ) Structure of phenoxazinone synthase from Streptomyces antibioticus reveals a new type 2 copper center. PMID : 16584173 : DOI : 10.1021/bi0525526 Abstract >>
The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.
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16. |
Yang M,
Proctor MR,
Bolam DN,
Errey JC,
Field RA,
Gilbert HJ,
Davis BG,
( 2005 ) Probing the breadth of macrolide glycosyltransferases: in vitro remodeling of a polyketide antibiotic creates active bacterial uptake and enhances potency. PMID : 15984838 : DOI : 10.1021/ja051482n Abstract >>
The glycan portion of macrolide antibiotics modulates their efficacy. High-level expression of three macrolide GTs and kinetic analysis has revealed a highly selective synthetic "tool kit" with such plasticity that 12 glycan-modified macrolide antibiotics have been readily created. One of these (1-Gal) is enhanced over its parent oleandomycin (1) by "glycotargeting", allowing higher uptake through active internalization by virtue of the attachment of a glycan (Gal) not normally found on 1. Subsequent release of the targeting glycan by endogenous galactosidase activity releases 1.
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17. |
Funa N,
Funabashi M,
Yoshimura E,
Horinouchi S,
( 2005 ) A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides. PMID : 15701630 : DOI : 10.1074/jbc.M500190200 Abstract >>
RppA is a type III polyketide synthase (PKS) that catalyzes condensation of five molecules of malonyl-CoA to form 1,3,6,8-tetrahydroxynaphthalene (THN). In Streptomyces antibioticus IFO13271 and several other Streptomyces species, an open reading frame, named momA, is present as a neighbor of rppA. MomA belonged to the "cupin" superfamily because it contained a set of two motifs that is responsible for binding one equivalent of metal ions. MomA catalyzed monooxygenation of the THN produced from malonyl-CoA by the action of RppA to form flaviolin. In addition, it used several polyketides as substrates and formed the corresponding quinones. MomA required redox-active transition metal ions (Ni(2+), Cu(2+), Fe(3+), Fe(2+), Mn(2+), and Co(2+)) for its activity, whereas it was inhibited by a redox-inert transition metal ion (Zn(2+)). MomA neither possessed any flavin prosthetic group nor required nicotinamide cofactors for monooxygenation, which shows that MomA as a member of the cupin superfamily is a novel monooxygenase. Consistent with the catalytic property of MomA, WhiE-ORFII showing similarity in amino acid sequence to MomA and containing a cupin domain also catalyzed monooxygenation of THN. whiE-ORFII is located immediately upstream of the "minimal PKS" gene within the whiE type II PKS gene cluster for biosynthesis of a gray spore pigment in Streptomyces coelicolor A3(2), and a number of whiE-ORFII homologues are present in the biosynthetic gene cluster for polyketides of type II in various Streptomyces species. These findings show that a novel class of quinone-forming monooxygenases is involved in modification of aromatic polyketides synthesized by PKSs of types II and III.
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18. |
Manteca A,
Kamphausen T,
Fanghanel J,
Fischer G,
Sanchez J,
( 2004 ) Cloning and characterization of a Streptomyces antibioticus ATCC11891 cyclophilin related to Gram negative bacteria cyclophilins. PMID : 15304318 : DOI : 10.1016/j.febslet.2004.06.091 Abstract >>
Cyclophilins are folding helper enzymes and represent a family of the enzyme class of peptidyl-prolyl cis-trans isomerases. Here, we report the molecular cloning and biochemical characterization of SanCyp18, an 18-kDa cyclophilin from Streptomyces antibioticus ATCC11891 located in the cytoplasm and constitutively expressed during development. Amino acid sequence analysis revealed a much higher homology to cyclophilins from Gram negative bacteria than to known cyclophilins from Streptomyces or other Gram positive bacteria. SanCyp18 is inhibited weakly by CsA, with a K(i) value of 21 microM, similar to cyclophilins from Gram negative bacteria. However, this value is more than 20-fold higher than the K(i) values reported for cyclophilins from other Gram positive bacteria, which makes SanCyp18 unique within this group. The presence of SanCyp18 in Streptomyces is likely due to horizontal gene transmission from Gram-negative bacteria to Streptomyces.
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19. |
Parra F,
Blanco G,
Martín Alonso JM,
Balbín M,
Méndez C,
Salas JA,
( 1992 ) Cloning and sequence of a gene encoding the L7/L12 ribosomal protein equivalent of Streptomyces antibioticus. PMID : 1511874 : DOI : 10.1016/0378-1119(92)90259-r Abstract >>
A 50S ribosomal(r) protein from the vegetative mycelium of Streptomyces antibioticus, that is absent or modified in the spore 50S r-subunit, was purified by HPLC. Determination of its N terminus and comparison with amino acid sequence data bases indicated a strong homology with the L7/L12 r-protein from Streptomyces griseus. Screening of a cosmid library of S. antibioticus chromosomal DNA with a 20-mer oligodeoxyribonucleotide probe, corresponding to an internal region of the N terminus, allowed the isolation of two hybridizing clones. A 0.90-kb HindIII-BamHI fragment from one of these clones was sequenced and found to contain a 387-bp open reading frame. The deduced gene product shows clear homology with L7/L12 r-protein equivalent from different bacteria.
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20. |
Smith AW,
Camara-Artigas A,
Olea C,
Francisco WA,
Allen JP,
( 2004 ) Crystallization and initial X-ray analysis of phenoxazinone synthase from Streptomyces antibioticus. PMID : 15272175 : DOI : 10.1107/S0907444904013204 Abstract >>
Phenoxazinone synthase, an oligomeric multicopper oxidase produced by Streptomyces antibioticus, is responsible for the six-electron oxidative coupling of two molecules of 4-methyl 3-hydroxyanthraniloyl pentapeptide to form the phenoxazinone chromophore of the antineoplastic agent actinomycin D. Spectroscopic studies have shown that the enzyme contains one type I (blue) and three to four type II copper centers. However, the exact arrangement of the copper centers in this multicopper oxidase is unknown. As a first step towards determining the three-dimensional structure of the enzyme, phenoxazinone synthase has been crystallized. The hexameric form of phenoxazinone synthase was purified from 72 h cultures of S. lividans containing the plasmid pIJ702. Purified hexamers were concentrated to 75 mg ml(-1) and used to grow two forms of crystals. Data collected from the two crystal forms were processed in two separate space groups. Crystals of both forms were grown at 288 K using the sitting-drop vapour-diffusion method. Native data sets extending to resolutions of 3.35 and 2.30 A have been collected and processed in space groups R32 and P1, respectively.
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21. |
Kim BJ,
Kim CJ,
Chun J,
Koh YH,
Lee SH,
Hyun JW,
Cha CY,
Kook YH,
( 2004 ) Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences. PMID : 15023980 : DOI : 10.1099/ijs.0.02941-0 Abstract >>
The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100 % similarity within the same species and 90.2-99.3 % similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.
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22. |
Le TB,
Stevenson CE,
Fiedler HP,
Maxwell A,
Lawson DM,
Buttner MJ,
( 2011 ) Structures of the TetR-like simocyclinone efflux pump repressor, SimR, and the mechanism of ligand-mediated derepression. PMID : 21354180 : DOI : 10.1016/j.jmb.2011.02.035 Abstract >>
Simocyclinone D8 (SD8), a potent DNA gyrase inhibitor made by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators. SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously, we have shown that the mature antibiotic SD8 or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of SD8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here, we report the crystal structures of SimR alone and in complex with either SD8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterized TetR-family transcriptional regulators: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of SD8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid-body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change.
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23. |
Le TB,
Schumacher MA,
Lawson DM,
Brennan RG,
Buttner MJ,
( 2011 ) The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding. PMID : 21835774 : DOI : 10.1093/nar/gkr640 PMC : PMC3241653 Abstract >>
SimR, a TetR-family transcriptional regulator (TFR), controls the export of simocyclinone, a potent DNA gyrase inhibitor made by Streptomyces antibioticus. Simocyclinone is exported by a specific efflux pump, SimX and the transcription of simX is repressed by SimR, which binds to two operators in the simR-simX intergenic region. The DNA-binding domain of SimR has a classical helix-turn-helix motif, but it also carries an arginine-rich N-terminal extension. Previous structural studies showed that the N-terminal extension is disordered in the absence of DNA. Here, we show that the N-terminal extension is sensitive to protease cleavage, but becomes protease resistant upon binding DNA. We demonstrate by deletion analysis that the extension contributes to DNA binding, and describe the crystal structure of SimR bound to its operator sequence, revealing that the N-terminal extension binds in the minor groove. In addition, SimR makes a number of sequence-specific contacts to the major groove via its helix-turn-helix motif. Bioinformatic analysis shows that an N-terminal extension rich in positively charged residues is a feature of the majority of TFRs. Comparison of the SimR-DNA and SimR-simocyclinone complexes reveals that the conformational changes associated with ligand-mediated derepression result primarily from rigid-body rotation of the subunits about the dimer interface.
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24. |
Le Roes-Hill M,
Goodwin C,
Burton S,
( 2009 ) Phenoxazinone synthase: what's in a name? PMID : 19268377 : DOI : 10.1016/j.tibtech.2009.01.001 Abstract >>
The name phenoxazinone synthase (PHS, 2-aminophenol:oxygen oxidoreductase, EC 1.10.3.4) is used for the enzyme catalysing the oxidative coupling of substituted o-aminophenols to produce phenoxazinones. This review reveals that the traditional classification of PHS conflicts with recent sequence-based information that shows its relationship with two distinct copper protein groups. Different PHS roles, namely spore pigmentation in Streptomyces antibioticus (phsA) and biosynthesis of the antibiotic grixazone in Streptomyces griseus subsp. griseus (GriF), indicate an example of convergent evolution. Here, we review the classification, distribution and roles of PHSs, comparing them with copper oxidases at genetic and structural levels and exploring their potential application in the production of new antibiotics.
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25. |
Wu P,
Wan D,
Xu G,
Wang G,
Ma H,
Wang T,
Gao Y,
Qi J,
Chen X,
Zhu J,
Li YQ,
Deng Z,
Chen W,
( 2017 ) An Unusual Protector-Prot?g? Strategy for the Biosynthesis of Purine Nucleoside Antibiotics. PMID : 28111097 : DOI : 10.1016/j.chembiol.2016.12.012 Abstract >>
Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that the biosynthesis of PTN and Ara-A employs an unusual protector-prot?g? strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions as a reversible oxidoreductase for the final step of PTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the host adenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.
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26. |
Lee YH,
Chen BF,
Wu SY,
Leu WM,
Lin JJ,
Chen CW,
Lo SC,
( 1988 ) A trans-acting gene is required for the phenotypic expression of a tyrosinase gene in Streptomyces. PMID : 2840357 : DOI : 10.1016/0378-1119(88)90418-0 Abstract >>
The melanin locus (melC) from Streptomyces antibioticus was previously shown to be composed of two open reading frames (ORFs), melC1 and melC2. The melC2 ORF codes for the polypeptide chain of tyrosinase (apotyrosinase). The function of melC1 is not known except that insertional mutation within it abolishes the tyrosinase activity. Here, we show that in Streptomyces lividans TK64 harboring melC1 mutated and melC2 intact (melC1- melC2+) plasmids, while there was no tyrosinase activity, melC transcript was synthesized and apotyrosinase could be detected. The apotyrosinase could be activated to a limited degree by incubation with copper ions, or by mixing the mycelial extract from a culture harboring a melC1- melC2+ (pPF950) plasmid with that from a culture containing a melC1+ melC2- (pSA1) plasmid. Complementation analysis showed that melC1 acted in trans on the tyrosinase gene expression. Together, these results suggest that melC1 encodes or regulates a copper-transfer protein serving an in vivo copper-donor function in the biosynthesis of active tyrosinase.
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27. |
Schäfer M,
Stevenson CEM,
Wilkinson B,
Lawson DM,
Buttner MJ,
( 2016 ) Substrate-Assisted Catalysis in Polyketide Reduction Proceeds via a Phenolate Intermediate. PMID : 27617849 : DOI : 10.1016/j.chembiol.2016.07.018 PMC : PMC5039031 Abstract >>
SimC7 is a polyketide ketoreductase involved in biosynthesis of the angucyclinone moiety of the gyrase inhibitor simocyclinone D8 (SD8). SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, catalyzes reduction of the C-7 carbonyl of the angucyclinone, and the resulting hydroxyl is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct mechanism. To investigate this possibility, we determined the structures of SimC7 alone, with NADP(+), and with NADP(+) and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad, including the active-site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active-site mutants, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate.
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28. |
Montemiglio LC,
Parisi G,
Scaglione A,
Sciara G,
Savino C,
Vallone B,
( 2016 ) Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis. PMID : 26475642 : DOI : 10.1016/j.bbagen.2015.10.009 Abstract >>
OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. Clotrimazole-bound OleP adopts an open form, held by a �k-�k stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C�VC bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.
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29. |
Barry CE,
Nayar PG,
Begley TP,
( 1989 ) Phenoxazinone synthase: mechanism for the formation of the phenoxazinone chromophore of actinomycin. PMID : 2477054 : DOI : 10.1021/bi00441a026 Abstract >>
Phenoxazinone synthase is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The mechanism of this complex 6-electron oxidation was determined by using a variety of substituted 2-aminophenols, designed to block the reaction at intermediate stages. Thus, with 3,5-di-tert-butyl-2-aminophenol as substrate, the reaction was blocked at the o-quinone imine 17; with 5-tert-butyl-2-aminophenol (19) as substrate, the reaction was blocked at the p-quinone imine 20; and with 5-methyl-2-aminophenol (21) as substrate, the reaction was blocked at the dihydro-2-aminophenoxazinone 22. These findings suggested a mechanism in which 2-aminophenoxazinone formation proceeded via a quinone imine intermediate 4 that was trapped by a second molecule of 2-aminophenol. Oxidation of the adduct 5 to the p-quinone imine 6 was followed by a second conjugate addition and a final 2-electron oxidation to give the product, 2-aminophenoxazinone. The role of the enzyme in the catalysis of each of these steps was examined. It was found that the second conjugate addition generated a racemic center at C4a, suggesting that this reaction did not occur at the active site. A deuterium isotope effect on the cleavage of the C4-H bond of 2-aminophenol suggested that partial dissociation of an intermediate from the enzyme occurred after the first conjugate addition. It is proposed that 2-aminophenoxazinone synthesis proceeds via a sequence of three consecutive 2-electron aminophenol oxidations and that the aminophenol moiety is regenerated during the reaction sequence by facile tautomerization reactions. Thus, what initially appears to be an impressively complex mechanism may, in fact, be ingeniously simple.
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30. |
Goomeshi Nobary S,
Jensen SE,
( 2012 ) A comparison of the clavam biosynthetic gene clusters in Streptomyces antibioticus T?1718 and Streptomyces clavuligerus. PMID : 22435762 : DOI : 10.1139/w2012-012 Abstract >>
The production of clavam metabolites has been studied previously in Streptomyces clavuligerus , a species that produces clavulanic acid as well as 4 other clavam compounds, but the late steps of the pathway leading to the specific end products are unclear. The present study compared the clavam biosynthetic gene cluster in Streptomyces antibioticus , chosen because it produces only 2 clavam metabolites and no clavulanic acid, with that of S. clavuligerus. A cosmid library of S. antibioticus genomic DNA was screened with a clavaminate synthase-specific probe based on the corresponding genes from S. clavuligerus, and 1 of the hybridizing cosmids was sequenced in full. A clavam gene cluster was identified that shows similarities to that of S. clavuligerus but also contains a number of novel genes. Knock-out mutation of the clavaminate synthase gene abolished clavam production in S. antibioticus, confirming the identity of the gene cluster. Knock-out mutation of a novel gene encoding an apparent oxidoreductase also abolished clavam production. A potential clavam biosynthetic pathway consistent with the genes in the cluster and the metabolites produced by S. antibioticus, and correspondingly different from that of S. clavuligerus, is proposed.
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31. |
Han JH,
Cho MH,
Kim SB,
( 2012 ) Ribosomal and protein coding gene based multigene phylogeny on the family Streptomycetaceae. PMID : 22154623 : DOI : 10.1016/j.syapm.2011.08.007 Abstract >>
The phylogenetic relationship among the three genera of the family Streptomycetaceae was examined using the small and large subunit ribosomal RNA genes, and the gyrB, rpoB, trpB, atpD and recA genes. The total stretches of the analyzed ribosomal genes were 4.2kb, and those of five protein coding genes were 4.5 kb. The resultant phylogenetic trees confirmed that each genus formed an independent clade in the majority of cases. The G+C contents of rRNA genes were 56.9-58.9 mol%, and those of protein coding genes were 65.4-72.4 mol%, the latter being closer to those of the genomic DNAs. The average nucleotide sequence identity between the organisms were 94.1-96.4% for rRNA genes and 85.7-90.6% for protein coding genes, thus indicating that protein coding genes can give higher resolution than rRNA genes. In addition, the protein coding gene trees were more stable than the rRNA gene trees, supported by higher bootstrap values and other treeing algorithms. Moreover, the genome data of six Streptomyces species indicated that many protein coding genes exhibited higher correlations with genome relatedness. The combined gene sequences were also shown to give a better resolution with higher stability than any single genes, though not necessarily more correlated with genome relatedness. It is evident from this study that the rRNA gene based phylogeny can be misleading, and also that protein coding genes have a number of advantages over the rRNA genes as the phylogenetic markers including a high correlation with the genome relatedness.
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32. |
( 1997 ) abaB, a putative regulator for secondary metabolism in Streptomyces. PMID : 9037760 : DOI : 10.1111/j.1574-6968.1997.tb10216.x Abstract >>
A chromosomal DNA fragment from Streptomyces antibioticus ATCC11891 was isolated by its ability to stimulate actinorhodin and undecylprodigiosin biosynthesis in Streptomyces lividans TK21. This fragment includes two open reading frames, whose deduced translated products resemble enzymes involved in sulfur metabolism (ORF1) and LysR-type transcriptional regulators (ORF2). The cloning of the promoter region of ORF2 (abaB) in high copy number led to overproduction of both antibiotics suggesting that this phenotype might well be due to titration by this region of one or more proteins. Southern blot analysis revealed that abaB gene is highly conserved among all streptomycetes tested.
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33. |
( 1996 ) Guanosine pentaphosphate synthetase from Streptomyces antibioticus is also a polynucleotide phosphorylase. PMID : 8763958 : DOI : 10.1128/jb.178.14.4281-4288.1996 PMC : PMC178187 Abstract >>
The gene for the enzyme guanosine pentaphosphate synthetase I (GPSI) from Streptomyces antibioticus has been cloned and sequenced. The cloned gene functioned as a template in the streptomycete coupled transcription-translation system and directed the synthesis of a protein with the properties expected for GPSI. Sequencing of the cloned gene identified an open reading frame of 740 amino acids whose amino terminal sequence corresponded to the N terminus of purified GPSI. The GPSI protein sequence was found to possess significant homology to polynucleotide phosphorylase from Escherichia coli. Indeed, like E. coli polynucleotide phosphorylase, purified GPSI was shown to catalyze the polymerization of ADP and the phosphorolysis of poly(A). However, the E. coli enzyme was unable to catalyze the synthesis of guanosine pentaphosphate under conditions in which GPSI was highly active in that reaction. Overexpression of the cloned gpsI gene in E. coli led to an increase in both polynucleotide phosphorylase and guanosine pentaphosphate synthetase activities in the cloning host. The polynucleotide phosphorylase activities of GPSI and of the E. coli enzyme were strongly inhibited by dCDP, but the pppGpp synthetase activity of GPSI was not inhibited and indeed was slightly stimulated by dCDP. These results strongly support the identity of GPSI as a bifunctional enzyme capable of both pppGpp synthesis and polynucleotide phosphorylase activities.
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34. |
( 1993 ) Characterization of a Streptomyces antibioticus gene cluster encoding a glycosyltransferase involved in oleandomycin inactivation. PMID : 8244027 : DOI : 10.1016/0378-1119(93)90189-a Abstract >>
By homology to the mgt gene (encoding a macrolide glycosyltransferase) from Streptomyces lividans, a 3.3-kb DNA fragment from the oleandomycin producer, Streptomyces antibioticus, was cloned and sequenced. Analysis of the sequence revealed the presence of the 3' end of a gene (ORF1) and two complete ORFs (ORF2 and oleD), all of them translationally coupled. The deduced product of the sequenced region of ORF1 contained the typical signature of integral membrane proteins responsible for the translocation of substrates across the membrane. The ORF2 product did not show significant similarity with proteins in databases, but contains an N-terminus leader peptide region characteristic of secreted proteins, and a lipid attachment site motif characteristic of membrane lipoproteins synthesized with a precursor signal peptide. The oleD product showed clear similarity with several UDP-glucuronosyl- and UDP-glycosyl-transferases from different origins and particularly with the mgt gene from S. lividans, and might encode a glycosyltransferase activity capable of inactivating macrolides. It is proposed that these three genes could participate in the intracellular glycosylation of oleandomycin and its secretion during antibiotic production.
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35. |
( 1997 ) Sigma-E is required for the production of the antibiotic actinomycin in Streptomyces antibioticus. PMID : 9004230 : DOI : 10.1046/j.1365-2958.1997.2001566.x Abstract >>
The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus. The phsA promoter strikingly resembles a putative Streptomyces sigma E cognate promoter, and purified E sigma E holoenzyme transcribed the phsA promoter in vitro. However, the phsA promoter was still active in an S. antibioticus sigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.
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36. |
( 1993 ) The unstable melC operon of Streptomyces antibioticus is codeleted with a Tn4811-homologous locus. PMID : 8383668 : DOI : 10.1128/jb.175.6.1847-1852.1993 PMC : PMC203993 Abstract >>
The melC operon of Streptomyces antibioticus is unstable, undergoing frequent spontaneous deletions. All the delta melC mutants analyzed also lost 2-kb V1 DNA, which contained two open reading frames (ORFs) homologous to ORF4 (a putative oxidoreductase gene) and ORF5 (a putative AraC-type regulatory gene) of Tn4811. The two ORFs may constitute an accessory unit of a different transposon.
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37. |
( 1993 ) Streptomyces antibioticus contains at least three oleandomycin-resistance determinants, one of which shows similarity with proteins of the ABC-transporter superfamily. PMID : 8326867 : DOI : 10.1111/j.1365-2958.1993.tb01601.x Abstract >>
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 transmembrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9 kb PstI-SphI 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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38. |
( 1994 ) Characterisation of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence. PMID : 8107683 : DOI : 10.1007/bf00280426 Abstract >>
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 bp 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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39. |
Iwasaki Y,
Nakano H,
Yamane T,
( 1994 ) Phospholipase D from Streptomyces antibioticus: cloning, sequencing, expression, and relationship to other phospholipases. PMID : 7765769 : Abstract >>
The extracellular phospholipase D (PLD) gene from Streptomyces antibioticus was cloned, sequenced, and expressed in Escherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form in E. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCl and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S. antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus and Streptomyces sp., and contained a conserved region with S. chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region XPLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.
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40. |
Janc JW,
Egan LA,
Townsend CA,
( 1995 ) Purification and characterization of clavaminate synthase from Streptomyces antibioticus. A multifunctional enzyme of clavam biosynthesis. PMID : 7890654 : DOI : 10.1074/jbc.270.10.5399 Abstract >>
Clavaminate synthase (CS), a key enzyme in the clavulanic acid biosynthetic pathway, has been purified to electrophoretic homogeneity from Streptomyces antibioticus (T? 1718), a species that does not produce clavulanic acid. A comparison of the physical and kinetic properties of clavaminate synthase from S. antibioticus (CS3) and the two isozymes from Streptomyces clavuligerus (CS1 and CS2) has been conducted. In oxidative reactions requiring the co-substrates O2, alpha-ketoglutaric acid, and catalytic Fe2+, both CS1 and CS2 catalyze three distinct transformations, the hydroxylation of deoxyguanidinoproclavaminic acid to guanidinoproclavaminic acid, and the cyclization and desaturation of proclavaminic acid to clavaminic acid. We have demonstrated that CS3 from S. antibioticus also catalyzes these three oxidations. The apparent molecular mass of CS3 from matrix-assisted laser desorption mass spectrometry is 35,839 +/- 36 Da. The enzyme is a monomer in solution as determined by gel filtration chromatography. Analysis of the four possible proclavaminic acid diastereomers confirmed the absolute configuration of the substrate to be 2S,3R. Based upon N-terminal sequence comparisons among the three proteins, CS3 possesses the higher degree of homology with the CS1 isozyme from S. clavuligerus. Although previously associated solely with clavulanic acid biosynthesis, we propose these findings and recent precursor incorporation data support the view that clavaminate synthase plays a critical role in the biosynthesis of the clavam metabolites.
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41. |
Hsieh CJ,
Jones GH,
( 1995 ) Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus. PMID : 7592317 : DOI : 10.1128/jb.177.20.5740-5747.1995 PMC : PMC177392 Abstract >>
The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.
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42. |
Rodriguez AM,
Olano C,
Méndez C,
Hutchinson CR,
Salas JA,
( 1995 ) A cytochrome P450-like gene possibly involved in oleandomycin biosynthesis by Streptomyces antibioticus. PMID : 7737473 : DOI : 10.1111/j.1574-6968.1995.tb07459.x Abstract >>
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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43. |
Golub EE,
Nishimura JS,
( 1972 ) Phenoxazinone synthetase from Streptomyces antibioticus: multiple activities of the enzyme. PMID : 4118295 : PMC : PMC251570 Abstract >>
A procedure for the preparation of relatively large quantities of highly purified phenoxazinone synthetase from Streptomyces antibioticus is described. Enzyme preparations consisted of multiple forms, as determined by polyacrylamide gel electrophoresis. Each of the electrophoretically separable forms catalyzed the oxidation of catechols, ferrocyanide, and ethylenic thiols, in addition to o-aminophenols.
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44. |
Bernan V,
Filpula D,
Herber W,
Bibb M,
Katz E,
( 1985 ) The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. PMID : 3932128 : DOI : 10.1016/0378-1119(85)90262-8 Abstract >>
The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.
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45. |
Choy HA,
Jones GH,
( 1981 ) Phenoxazinone synthase from Streptomyces antibiotics: purification of the large and small enzyme forms. PMID : 7305384 : DOI : 10.1016/0003-9861(81)90429-x Abstract >>
N/A
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46. |
Semsary S,
Crnov?i? I,
Driller R,
Vater J,
Loll B,
Keller U,
( 2018 ) Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4-Oxoproline Synthase. PMID : 29327817 : DOI : 10.1002/cbic.201700666 Abstract >>
X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X0) or 4-oxoproline (Acm X2) in their �]-pentapeptide lactone rings, whereas their �\ ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we show-using an artificial Acm half analogue (PPL 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4''-oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.
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47. |
Zhang B,
Tian W,
Wang S,
Yan X,
Jia X,
Pierens GK,
Chen W,
Ma H,
Deng Z,
Qu X,
( 2017 ) Activation of Natural Products Biosynthetic Pathways via a Protein Modification Level Regulation. PMID : 28562006 : DOI : 10.1021/acschembio.7b00225 Abstract >>
Natural products are critical for drug discovery and development; however their discovery is challenged by the wide inactivation or silence of microbial biosynthetic pathways. Currently strategies targeting this problem are mainly concentrated on chromosome dissembling, transcription, and translation-stage regulations as well as chemical stimulation. In this study, we developed a novel approach to awake cryptic/silenced microbial biosynthetic pathways through augmentation of the conserved protein modification step-phosphopantetheinylation of carrier proteins. Overexpression of phosphopantetheinyl transferase (Pptase) genes into 33 Actinomycetes achieved a significantly high activation ratio at which 23 (70%) strains produced new metabolites. Genetic and biochemical studies on the mode-of-action revealed that exogenous PPtases triggered the activation of carrier proteins and subsequent production of metabolites. With this approach we successfully identified five oviedomycin and halichomycin-like compounds from two strains. This study provides a novel approach to efficiently activate cryptic/silenced biosynthetic pathways which will be useful for natural products discovery.
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48. |
Parisi G,
Montemiglio LC,
Giuffrè A,
Macone A,
Scaglione A,
Cerutti G,
Exertier C,
Savino C,
Vallone B,
( 2019 ) Substrate-induced conformational change in cytochrome P450 OleP. PMID : 30207799 : DOI : 10.1096/fj.201800450RR Abstract >>
The regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.-Parisi, G., Montemiglio, L. C., Giuffr?, A., Macone, A., Scaglione, A., Cerutti, G., Exertier, C., Savino, C., Vallone, B. Substrate-induced conformational change in cytochrome P450 OleP.
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( 1998 ) Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus. PMID : 9680207 : DOI : 10.1046/j.1365-2958.1998.00880.x Abstract >>
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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( 1998 ) Comparative ribosomal protein (L11 and L30) sequence analyses of several Streptomyces spp. commonly used in genetic studies. PMID : 9731302 : DOI : 10.1099/00207713-48-2-597 Abstract >>
The taxonomic relationships among nine strains of Streptomyces, which have been commonly used for genetic studies, were examined by sequence analysis of their ribosomal L11(= rplK) protein genes. Phylogenetic relationships among these organisms derived from similarity sequence analysis of the rplK genes were in good agreement with those derived from the analysis of the deduced L11 protein amino acid sequence itself, indicating complete sequence homology among Streptomyces coelicolor A3(2), 'Streptomyces lividans 66' and Streptomyces violaceoruber JCM 4423. S. coelicolor A3(2) related (in the order of closer relatedness) to Streptomyces antibioticus ATCC 14888, Streptomyces griseus IFO 13189, Streptomyces lavendulae MA 406 A-1 and Streptomyces virginiae MAFF 6014. Sequence analysis of the 26 N-terminal amino acid residues of ribosomal L30 proteins also resulted in similar phylogenetic relationships, except that S. griseus, S. lavendulae and S. virginiae were not differentiated from each other using this method. These findings concerning the phylogenetic relationship therefore confirm the previous conclusion that S. coelicolor A3(2), 'S. lividans 66' and S. violaceoruber should be recognized as a single taxon at the species level.
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( 1998 ) Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring. PMID : 9749673 : DOI : 10.1007/s004380050816 Abstract >>
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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( 1998 ) Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus. PMID : 9518550 : DOI : 10.1016/s0005-2760(97)00191-4 Abstract >>
Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host. The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation. Both of them were confirmed to be expressed in S. antibioticus. The two enzymes were different in the following properties. (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes. (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not. (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them. (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have. Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate. Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues. PLC1 has residues that possibly form a Ca2+-binding site. Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes. Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes.
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