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1. Forsman  M, Granström  M,     ( 1992 )

Mutagenic analysis of the promoter of the Streptomyces fradiae beta-lactamase-encoding gene.

Gene 121 (1)
PMID : 1385267  :   DOI  :   10.1016/0378-1119(92)90165-l    
Abstract >>
The Streptomyces fradiae beta-lactamase promoter (PblaF) was sequenced and characterized by promoter probing, primer extension, and exonuclease III-mediated deletions. The transcription start point (tsp) was the same in both S. lividans and S. fradiae. Oligodeoxyribonucleotide-directed random mutations and site-specific mutations were introduced in the promoter region. The effects of these mutations on transcription were assayed by an RNA colony hybridization method. This analysis identified cis-acting sequence determinants located similarly to the -10 and -35 regions of a typical Escherichia coli promoter. Also, a change in the distance between these regions from 19 to 17 bp drastically reduced promoter activity. PblaF was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC, or hrdD. Sequence alignment of PblaF to sigma factor-classified Streptomyces promoters revealed little homology. Thus, PblaF is probably recognized by an as yet unidentified sigma factor.
KeywordMeSH Terms
Promoter Regions, Genetic
2. Tamegai  H, Eguchi  T, Kakinuma  K,     ( 2002 )

First identification of Streptomyces genes involved in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics--genetic and evolutionary analysis of L-glutamine:2-deoxy-scyllo-inosose aminotransferase genes.

The Journal of antibiotics 55 (11)
PMID : 12546424  :   DOI  :   10.7164/antibiotics.55.1016    
Abstract >>
N/A
KeywordMeSH Terms
3. Chen  H, Yamase  H, Murakami  K, Chang  CW, Zhao  L, Zhao  Z, Liu  HW,     ( 2002 )

Expression, purification, and characterization of two N,N-dimethyltransferases, tylM1 and desVI, involved in the biosynthesis of mycaminose and desosamine.

Biochemistry 41 (29)
PMID : 12119032  :   DOI  :   10.1021/bi020245j    
Abstract >>
Methylation catalyzed by an S-adenosylmethionine- (AdoMet-) dependent methyltransferase is an effective means to alter the hydrophilicity and/or nucleophilicity of a molecule. While a large number of enzymes capable of catalyzing methylation at carbon, oxygen, sulfur, and nitrogen atoms are known, only a few are able to catalyze N,N-dimethylation. Mycaminose and desosamine are aminohexoses found in several macrolide antibiotics, such as tylosin and methymycin, respectively. Both sugars contain a C-3 N,N-dimethylamino group which has been shown to confer the biological activity of these unusual sugars. Recently, sequence analysis as well as genetic studies has led to the assignment of tylM1 in the tylosin biosynthetic gene cluster and desVI in the methymycin biosynthetic gene cluster as genes encoding the corresponding N,N-dimethyltransferases. To verify the proposed roles of the tylM1 and desVI genes, we have overexpressed and purified their encoded products, synthesized the predicted substrates, and characterized the catalytic function of these proteins. Our studies showed that TylM1 and DesVI are homodimeric proteins and have nearly identical biochemical properties. These enzymes do not have strong preference for binding either the unmethylated substrate or the monomethylated intermediate. It is the chemical reactivity of the nitrogen functional group that determines the relative rate of a particular methylation step. Thus, our results not only establish TylM1 and DesVI as new members of a small family of enzymes that are capable of catalyzing N,N-dimethylation of an amino group but also provide evidence indicating that the methylation catalyzed by AdoMet-dependent methyltransferases proceeds in a stepwise manner and is nucleophilic in nature.
KeywordMeSH Terms
4. Butler  AR, Gandecha  AR, Cundliffe  E,     ( 2001 )

Influence of ancillary genes, encoding aspects of methionine metabolism, on tylosin biosynthesis in Streptomyces fradiae.

The Journal of antibiotics 54 (8)
PMID : 11594346  :   DOI  :   10.7164/antibiotics.54.642    
Abstract >>
The tylosin-biosynthetic (tyl) gene cluster of Streptomyces fradiae contains ancillary genes that encode functions normally associated with primary metabolism. These can be disrupted without loss of viability, since equivalent genes (presumably used for 'housekeeping' purposes) are also present elsewhere in the genome. The tyl cluster also contains two genes that encode products unlike any proteins in the databases. Two ancillary genes, metF (encoding N5,N10-methylenetetrahydrofolate reductase) and metK, encoding S-adenosylmethionine synthase, flank one of the 'unknown' genes (orf9) in the tyl cluster. In a strain of S. fradiae in which all three of these genes were disrupted, tylosin production was reduced, although this effect was obscured in media supplemented with glycine betaine which can donate methyl groups to the tetrahydrofolate pool. Apparently, one consequence of the recruitment of ancillary genes into the tyl cluster is enhanced capacity for transmethylation during secondary metabolism.
KeywordMeSH Terms
5. Hoffmeister  D, Ichinose  K, Domann  S, Faust  B, Trefzer  A, Dräger  G, Kirschning  A, Fischer  C, Künzel  E, Bearden  D, Rohr  J, Bechthold  A,     ( 2000 )

The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster.

Chemistry & biology 7 (11)
PMID : 11094336  :  
Abstract >>
Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.
KeywordMeSH Terms
6. Liu  M, Kirpekar  F, Van Wezel  GP, Douthwaite  S,     ( 2000 )

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA.

Molecular microbiology 37 (4)
PMID : 10972803  :   DOI  :   10.1046/j.1365-2958.2000.02046.x    
Abstract >>
tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.
KeywordMeSH Terms
7. Schneider  P, Decker  H, Haag  S, Westrich  L, Weitnauer  G, Hoffmeister  D, Faust  B,     ( 2000 )

Two new tailoring enzymes, a glycosyltransferase and an oxygenase, involved in biosynthesis of the angucycline antibiotic urdamycin A in Streptomyces fradiae T?2717.

Microbiology (Reading, England) 146 (Pt 1) (N/A)
PMID : 10658661  :   DOI  :   10.1099/00221287-146-1-147    
Abstract >>
Urdamycin A, the principal product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked D-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 3.35 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZl, urdGT2, urdG and urdH) on an 8.05 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C-C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, the C-glycosyltransfer of one NDP-D-olivose.
KeywordMeSH Terms
8. Butler  AR, Smith  IP, Bate  N,     ( 2000 )

The mycarose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.

Microbiology (Reading, England) 146 (Pt 1) (N/A)
PMID : 10658660  :   DOI  :   10.1099/00221287-146-1-139    
Abstract >>
The tylCK region of the Streptomyces fradiae genome was sequenced, revealing an incomplete set of five tylC genes encoding all-but-one of the enzymes involved in the biosynthesis of mycarose. The latter is a 6-deoxyhexose sugar required during production of the macrolide antibiotic, tylosin. The missing mycarose-biosynthetic gene, tylCVI, was found about 50 kb distant from its functional partners, on the other side of the tylG (polyketide synthase) gene complex. Mutational analysis, involving targeted gene transplacement, was employed to confirm the functions of specific genes, including tylCVI. Particularly interesting was the similarity between the tylosin-biosynthetic mycarosyltransferase enzyme, TylCV, and proteins of the macrolide glycosyltransferase (MGT) family that inactivate macrolides via glycosylation of attached sugar residues and are involved in resistance and/or antibiotic efflux. The arrangement of genes within the 'mycarose cluster' would allow their expression as two short operons with divergent, and perhaps co-regulated, promoters. Whether displacement of tylCVI relative to the other tylC genes provides additional regulatory opportunities remains to be established.
KeywordMeSH Terms
9. Bate  N,     ( 1999 )

The mycinose-biosynthetic genes of Streptomyces fradiae, producer of tylosin.

Journal of industrial microbiology & biotechnology 23 (2)
PMID : 10510490  :  
Abstract >>
The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl.
KeywordMeSH Terms
10. Gandecha  AR, Butler  AR,     ( 1999 )

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae.

Chemistry & biology 6 (9)
PMID : 10467127  :  
Abstract >>
The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.
KeywordMeSH Terms
11. Cundliffe  E,     ( 1999 )

Molecular analysis of tlrB, an antibiotic-resistance gene from tylosin-producing Streptomyces fradiae, and discovery of a novel resistance mechanism.

The Journal of antibiotics 52 (3)
PMID : 10348045  :   DOI  :   10.7164/antibiotics.52.288    
Abstract >>
The tlrB gene, which confers inducible resistance to a range of macrolide antibiotics including biosynthetic precursors of tylosin, was isolated and sequenced. In the genome of Streptomyces fradiae, it lies between pbp, which encodes a putative penicillin-binding protein, and tylN, encoding a glycosyltransferase involved in tylosin biosynthesis. The TlrB protein was produced in E. coli as a fusion to MalE. The fusion protein, but not MalE alone, inactivates macrolides in the presence of S-adenosyl-methionine (SAM) but the modified product(s) has not been characterised.
KeywordMeSH Terms
ATP-Binding Cassette Transporters
Escherichia coli Proteins
Genes, Bacterial
Monosaccharide Transport Proteins
Periplasmic Binding Proteins
12. Fouces  R, Barredo  JL, Díez  B,     ( 1999 )

The tylosin biosynthetic cluster from Streptomyces fradiae: genetic organization of the left region.

Microbiology (Reading, England) 145 (Pt 4) (N/A)
PMID : 10220165  :   DOI  :   10.1099/13500872-145-4-855    
Abstract >>
The genetic organization of the left edge (tyIEDHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, ddcA), tylosin resistance determinant (ORF2, tlrB), glycosyltransferase (ORF3, tylN), methyltransferase (ORF4, tylE), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH1), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked at various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-D-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed.
KeywordMeSH Terms
Genes, Bacterial
13. Sinha  U, Wolz  SA, Lad  PJ,     ( 1991 )

Two new extracellular serine proteases from Streptomyces fradiae.

The International journal of biochemistry 23 (10)
PMID : 1786859  :   DOI  :   10.1016/0020-711x(91)90133-8    
Abstract >>
1. Two new extracellular serine proteases have been purified to homogeneity from Streptomyces fradiae. 2. On amino acid sequencing, striking homology is observed between the first enzyme and Streptomyces griseus Protease A, and the second enzyme and S. griseus trypsin. 3. The sequence information shows for the first time that structurally and enzymatically related serine proteases are extracellularly expressed by different Streptomycetes. 4. Differential keratinolytic substrate specificity among these two microbes are probable due to a difference in disulfide reduction capacity.
KeywordMeSH Terms
14. Li  J, Shi  PJ, Han  XY, Meng  K, Yang  PL, Wang  YR, Luo  HY, Wu  NF, Yao  B, Fan  YL,     ( 2007 )

Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris.

Protein expression and purification 54 (1)
PMID : 17408967  :   DOI  :   10.1016/j.pep.2007.02.012    
Abstract >>
We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.
KeywordMeSH Terms
15. Keatinge-Clay  AT,     ( 2007 )

A tylosin ketoreductase reveals how chirality is determined in polyketides.

Chemistry & biology 14 (8)
PMID : 17719489  :   DOI  :   10.1016/j.chembiol.2007.07.009    
Abstract >>
Because it controls the majority of polyketide stereocenters, the ketoreductase (KR) is a central target in engineering polyketide synthases (PKSs). To elucidate the mechanisms of stereocontrol, the structure of KR from the first module of the tylosin PKS was determined. A comparison with a recently solved erythromycin KR that operates on the same substrate explains why their products have opposite alpha-substituent chiralities. The structure reveals how polyketides are guided into the active site by key residues in different KR types. There are four types of reductase-competent KRs, each capable of fixing a unique combination of alpha-substituent and beta-hydroxyl group chiralities, as well as two types of reductase-incompetent KRs that control alpha-substituent chirality alone. A protocol to assign how a module will enforce substituent chirality based on its sequence is presented.
KeywordMeSH Terms
16. Woodyer  RD, Li  G, Zhao  H, van der Donk  WA,     ( 2007 )

New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin.

Chemical communications (Cambridge, England) N/A (4)
PMID : 17220970  :   DOI  :   10.1039/b614678c    
Abstract >>
Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.
KeywordMeSH Terms
17. Woodyer  RD, Shao  Z, Thomas  PM, Kelleher  NL, Blodgett  JA, Metcalf  WW, van der Donk  WA, Zhao  H,     ( 2006 )

Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster.

Chemistry & biology 13 (11)
PMID : 17113999  :   DOI  :   10.1016/j.chembiol.2006.09.007    
Abstract >>
Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.
KeywordMeSH Terms
Multigene Family
18. Huang  F, Spiteller  D, Koorbanally  NA, Li  Y, Llewellyn  NM, Spencer  JB,     ( 2007 )

Elaboration of neosamine rings in the biosynthesis of neomycin and butirosin.

Chembiochem : a European journal of chemical biology 8 (3)
PMID : 17206729  :   DOI  :   10.1002/cbic.200600371    
Abstract >>
The proteins Neo-11 and Neo-18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl-6'-oxidase and 6'-oxoglucosaminyl:L-glutamate aminotransferase, respectively. The joint activity of Neo-11 and Neo-18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD-dependent dehydrogenation followed by a pyridoxal-5'-phosphate-mediated transamination. Neo-18 is also shown to catalyze deamination at C-6''' of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin. The product of the btrB gene, a homologue of neo-18 in the butirosin biosynthetic gene cluster (btr) in Bacillus circulans, exhibits the same activity as Neo-18; this indicates that there is a similar reaction sequence in both butirosin and neomycin biosynthesis.
KeywordMeSH Terms
19. Liu  XQ, Yang  XQ, Xie  FH, Song  LY, Zhang  GQ, Qian  SJ,     ( 2007 )

On-column refolding and purification of transglutaminase from Streptomyces fradiae expressed as inclusion bodies in Escherichia coli.

Protein expression and purification 51 (2)
PMID : 16935525  :   DOI  :   10.1016/j.pep.2006.07.011    
Abstract >>
Since transglutaminase (TGase) have been widely used in industry, mass production of the enzyme is especially necessary. The mature TGase gene from Streptomyces fradiae was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3). The recombinant TGase was formed as inclusion bodies, and its content was as high as 55% of the total protein content. The insoluble fractions were separated from cellular debris by centrifugation and solubilized with 8 M urea. With an on-column refolding procedure based on cation SP Fast Flow chromatography with dual-gradient, the active TGase protein was recovered efficiently from inclusion bodies. The final purified product was 95% pure detected by SDS-PAGE. Under appropriate experimental conditions, the protein yield and specific activity of the TGase were up to 53% and 21 U/mg, respectively. Furthermore, the refolded recombinant protein demonstrated nearly identical ability to polymerized BSA compared with that of native TGase. One hundred and five milligrams of refolded TGase protein was obtained from 3.2g wet weight cells in the 400 ml cell culture.
KeywordMeSH Terms
20. Kudo  F, Yamamoto  Y, Yokoyama  K, Eguchi  T, Kakinuma  K,     ( 2005 )

Biosynthesis of 2-deoxystreptamine by three crucial enzymes in Streptomyces fradiae NBRC 12773.

The Journal of antibiotics 58 (12)
PMID : 16506694  :   DOI  :   10.1038/ja.2005.104    
Abstract >>
NeoA, B, and C encoded in the neomycin biosynthetic gene cluster have been enzymatically confirmed to be responsible to the formation of 2-deoxystreptamine (DOS) in Streptomyces fradiae. NeoC was functionally characterized as 2-deoxy-scyllo-inosose synthase, which catalyzes the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. Further, NeoA appeared to catalyze the oxidation of 2-deoxy-scyllo-inosamine (DOIA) with NAD(P)+ forming 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Consequently, NeoA was characterized as 2-deoxy-scyllo-inosamine dehydrogenase. Finally, amino-DOI produced by NeoA from DOIA was transformed into DOS by NeoB. Since NeoB (Neo6) was also reported to be L-glutamine:2-deoxy-scyllo-inosose aminotransferase, all the enzymes in the DOS biosynthesis were characterized for the first time.
KeywordMeSH Terms
21. Miao  V, Brost  R, Chapple  J, She  K, Gal  MF, Baltz  RH,     ( 2006 )

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae.

Journal of industrial microbiology & biotechnology 33 (2)
PMID : 16208464  :   DOI  :   10.1007/s10295-005-0028-5    
Abstract >>
Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.
KeywordMeSH Terms
Multigene Family
22. Huang  F, Haydock  SF, Mironenko  T, Spiteller  D, Li  Y, Spencer  JB,     ( 2005 )

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: characterisation of an aminotransferase involved in the formation of 2-deoxystreptamine.

Organic & biomolecular chemistry 3 (8)
PMID : 15827636  :   DOI  :   10.1039/b501199j    
Abstract >>
The biosynthetic gene cluster of the 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic neomycin has been cloned for the first time by screening of a cosmid library of Streptomyces fradiae NCIMB 8233. Sequence analysis has identified 21 putative open reading frames (ORFs) in the neomycin gene cluster (neo) with significant protein sequence similarity to gene products involved in the biosynthesis of other DOS-containing aminoglycosides, namely butirosin (btr), gentamycin (gnt), tobramycin (tbm) and kanamycin (kan). Located at the 5'-end of the neo gene cluster is the previously-characterised neomycin phosphotransferase gene (apH). Three genes unique to the neo and btr clusters have been revealed by comparison of the neo cluster to btr, gnt, tbm and kan clusters. This suggests that these three genes may be involved in the transfer of a ribose moiety to the DOS ring during the antibiotic biosynthesis. The product of the neo-6 gene is characterised here as the L-glutamine : 2-deoxy-scyllo-inosose aminotransferase responsible for the first transamination in DOS biosynthesis, which supports the assignment of the gene cluster.
KeywordMeSH Terms
23. Romero  NM, Parro  V, Malpartida  F, Mellado  RP,     ( 1992 )

Heterologous activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.

Nucleic acids research 20 (11)
PMID : 1614864  :   DOI  :   10.1093/nar/20.11.2767     PMC  :   PMC336920    
Abstract >>
A DNA fragment of Streptomyces fradiae is able to activate the antibiotic actinorhodin biosynthetic pathway when cloned in Streptomyces lividans. The activator DNA region has been sequenced and its transcription initiation and termination sites accurately mapped in vivo. This DNA encodes a 132 nucleotides long transcript which is apparently responsible for the actinorhodin production phenotype, possibly acting as an antisense RNA. The sequence of the activator gene revealed no homology with any other known Streptomyces coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
24. Melançon  CE, Takahashi  H, Liu  HW,     ( 2004 )

Characterization of tylM3/tylM2 and mydC/mycB pairs required for efficient glycosyltransfer in macrolide antibiotic biosynthesis.

Journal of the American Chemical Society 126 (51)
PMID : 15612702  :   DOI  :   10.1021/ja043900e    
Abstract >>
The heterologous expression of tylM3 and mydC, two homologous genes of previously unknown function, along with genes encoding their respective partner glycosyltransferases, tylM2 and mycB, and the necessary sugar biosynthesis genes significantly enhances the glycosyltransferase activity in the engineered Streptomyces venezuelae host in which the native glycosyltransferase, desVII, has been inactivated. Both glycosyltransferases accept the endogenous 12-membered macrolide, 10-deoxymethynolide, or the exogenously fed 16-membered macrolide, tylactone. Five new compounds were generated using this expression system. This work suggests that the 13 other known TylM3/MydC/DesVIII homologues found in macrolide and anthracycline antibiotic clusters likely function as glycosyltransferase auxiliary proteins as well. These findings will greatly assist endeavors to generate new natural products in these pathways in a combinatorial fashion.
KeywordMeSH Terms
25. Douthwaite  S, Crain  PF, Liu  M, Poehlsgaard  J,     ( 2004 )

The tylosin-resistance methyltransferase RlmA(II) (TlrB) modifies the N-1 position of 23S rRNA nucleotide G748.

Journal of molecular biology 337 (5)
PMID : 15046978  :   DOI  :   10.1016/j.jmb.2004.02.030    
Abstract >>
The methyltransferase RlmA(II) (TlrB) confers resistance to the macrolide antibiotic tylosin in the drug-producing strain Streptomyces fradiae. The resistance conferred by RlmA(II) is highly specific for tylosin, and no resistance is conferred to other macrolide drugs, or to lincosamide and streptogramin B (MLS(B)) drugs that bind to the same region on the bacterial ribosome. In this study, the methylation site of RlmA(II) is identified unambiguously by liquid chromatography/electrospray ionization mass spectrometry as the N-1 position of 23S rRNA nucleotide G748. This position is contacted by the mycinose sugar moiety of tylosin, which is absent from the other drugs. The selective resistance to tylosin conferred by m(1)G748 illustrates how differences in drug structure facilitate the drug fit at the MLS(B)-binding site. This observation is of relevance for the rational design of novel antimicrobials targeting the MLS(B) site, especially if the antimicrobials are to be used against pathogens possessing m(1)G748.
KeywordMeSH Terms
Drug Resistance, Microbial
26. 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.

International journal of systematic and evolutionary microbiology 54 (Pt 2)
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.
KeywordMeSH Terms
27. Carney  AE, Holden  HM,     ( 2011 )

Molecular architecture of TylM1 from Streptomyces fradiae: an N,N-dimethyltransferase involved in the production of dTDP-D-mycaminose.

Biochemistry 50 (5)
PMID : 21142177  :   DOI  :   10.1021/bi101733y     PMC  :   PMC3079244    
Abstract >>
d-Mycaminose is an unusual dideoxy sugar found attached to the antibiotic tylosin, a commonly used veterinarian therapeutic. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar. The last step in its biosynthesis involves the dimethylation of the hexose C-3' amino group by an S-adenosylmethionine (SAM) dependent enzyme referred to as TylM1. Here we report two high-resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosylhomocysteine (SAH) and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 and 1.79 ? resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-3' amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of SAM. Both Tyr 14 and Arg 241 serve to anchor the dTDP-linked sugar to the protein. To test the role of His 123 in catalysis, two site-directed mutant proteins were constructed, H123A and H123N. Both mutant proteins retained catalytic activity, albeit with reduced rates. Specifically, the k(cat)/K(m) was reduced to 1.8% and 0.37% for the H123A and H123N mutant proteins, respectively. High-resolution X-ray models showed that the observed perturbations in the kinetic constants were not due to major changes in their three-dimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds.
KeywordMeSH Terms
28. Salauze  D, Perez-Gonzalez  JA, Piepersberg  W, Davies  J,     ( 1991 )

Characterisation of aminoglycoside acetyltransferase-encoding genes of neomycin-producing Micromonospora chalcea and Streptomyces fradiae.

Gene 101 (1)
PMID : 2060791  :   DOI  :   10.1016/0378-1119(91)90237-6    
Abstract >>
Two genes (aac) encoding aminoglycoside-N-acetyltransferase from Streptomyces fradiae and Micromonospora chalcea were cloned: the former identified by hybridization with a homologous gene from Streptomyces rimosus forma paromomycinus, the second by direct expression in Streptomyces lividans using pIJ702 as a vector. These two genes showed pronounced nucleotide and amino acid sequence similarities between themselves and also between previously described streptomycetes aac genes. Comparison of the flanking sequence of actinomycetes aac genes indicates considerable divergence, contrary to the notion that clustered biosynthetic genes for structurally related antibiotics were disseminated in their entirety between microbial species.
KeywordMeSH Terms
Genes, Bacterial
29. Alexander  DC, Rock  J, He  X, Brian  P, Miao  V, Baltz  RH,     ( 2010 )

Development of a genetic system for combinatorial biosynthesis of lipopeptides in Streptomyces fradiae and heterologous expression of the A54145 biosynthesis gene cluster.

Applied and environmental microbiology 76 (20)
PMID : 20802082  :   DOI  :   10.1128/AEM.01248-10     PMC  :   PMC2953024    
Abstract >>
A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the �pC31 or �pBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu??), at the expense of factors containing 3-methyl-glutamic acid (3mGlu??). This provided a practical route to produce high levels of pure Glu??-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.
KeywordMeSH Terms
Multigene Family
30. Rosteck  PR, Reynolds  PA, Hershberger  CL,     ( 1991 )

Homology between proteins controlling Streptomyces fradiae tylosin resistance and ATP-binding transport.

Gene 102 (1)
PMID : 1864505  :   DOI  :   10.1016/0378-1119(91)90533-h    
Abstract >>
A tylosin(Ty)-producing strain of Streptomyces fradiae contains at least three genes, tlrA, tlrB, tlrC, specifying resistance to Ty (TyR). The complete nucleotide sequence of the TyR-encoding gene, tlrC, and the transcription start point of the gene were determined. The sequence contains an open reading frame coding for a protein of 548 amino acids (aa) with an Mr of 59129. The TlrC protein was identified by expression of the cloned gene by in vitro coupled transcription and translation in cell-free extracts derived from Streptomyces lividans. The N- and C-terminal halves of TlrC share extensive homology, suggesting that the protein evolved through tandem gene duplication. Each half of the deduced TlrC aa sequence also shows significant homology to numerous eukaryotic and prokaryotic membrane-associated, active-transport protein subunits. The homologous proteins include examples from the systems responsible for efflux of cytotoxic drugs from multidrug-resistant human cells and for export of hemolysin from Escherichia coli. The greatest similarity to TlrC is in regions containing the ATP-binding sites found in these proteins. These results suggest a role for the tlrC gene product as part of a multiple component, ATP-dependent transport system for the active excretion of Ty from the producing organism.
KeywordMeSH Terms
31. Yokoyama  K, Yamamoto  Y, Kudo  F, Eguchi  T,     ( 2008 )

Involvement of two distinct N-acetylglucosaminyltransferases and a dual-function deacetylase in neomycin biosynthesis.

Chembiochem : a European journal of chemical biology 9 (6)
PMID : 18311744  :   DOI  :   10.1002/cbic.200700717    
Abstract >>
N/A
KeywordMeSH Terms
32. Li  N, Yang  P, Wang  Y, Luo  H, Meng  K, Wu  N, Fan  Y, Yao  B,     ( 2008 )

Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. k11.

Journal of microbiology and biotechnology 18 (3)
PMID : 18388456  :  
Abstract >>
The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437 bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-beta-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and 60 degrees , respectively. The enzyme showed stability over a pH range of 4-10. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by Fe2+ and strongly inhibited by Hg2+ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.
KeywordMeSH Terms
Cloning, Molecular
Gene Expression
33. Mittler  M, Bechthold  A, Schulz  GE,     ( 2007 )

Structure and action of the C-C bond-forming glycosyltransferase UrdGT2 involved in the biosynthesis of the antibiotic urdamycin.

Journal of molecular biology 372 (1)
PMID : 17640665  :   DOI  :   10.1016/j.jmb.2007.06.005    
Abstract >>
The glycosyltransferase UrdGT2 from Streptomyces fradiae catalyzes the formation of a glycosidic C-C bond between a polyketide aglycone and D-olivose. The enyzme was expressed in Escherichia coli, purified and crystallized. Its structure was established by X-ray diffraction at 1.9 A resolution. It is the first structure of a C-glycosyltransferase. UrdGT2 belongs to the structural family GT-B of the glycosyltransferases and is likely to form a C(2)-symmetric dimer in solution. The binding structures of donor and acceptor substrates in five structurally homologous enzymes provided a clear and consistent guide for the substrate-binding structure in UrdGT2. The modeled substrate locations suggest the deeply buried Asp137 as the activator for C-C bond formation and explain the reaction. The putative model can be used to design mutations that change the substrate specificity. Such mutants are of great interest in overcoming the increasing danger of antibiotic resistance.
KeywordMeSH Terms
34. Tam  HK, Härle  J, Gerhardt  S, Rohr  J, Wang  G, Thorson  JS, Bigot  A, Lutterbeck  M, Seiche  W, Breit  B, Bechthold  A, Einsle  O,     ( 2015 )

Structural characterization of O- and C-glycosylating variants of the landomycin glycosyltransferase LanGT2.

Angewandte Chemie (International ed. in English) 54 (9)
PMID : 25581707  :   DOI  :   10.1002/anie.201409792     PMC  :   PMC4376353    
Abstract >>
The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates.
KeywordMeSH Terms
C-glycosylation
Friedel-Crafts alkylation
carbasugars
enzyme engineering
glycosyltransferases
C-glycosylation
Friedel-Crafts alkylation
carbasugars
enzyme engineering
glycosyltransferases
35. Patrikainen  P, Niiranen  L, Thapa  K, Paananen  P, Tähtinen  P, Mäntsälä  P, Niemi  J, Metsä-Ketelä  M,     ( 2014 )

Structure-based engineering of angucyclinone 6-ketoreductases.

Chemistry & biology 21 (10)
PMID : 25200607  :   DOI  :   10.1016/j.chembiol.2014.07.017    
Abstract >>
Angucyclines are tetracyclic polyketides produced by Streptomyces bacteria that exhibit notable biological activities. The great diversity of angucyclinones is generated in tailoring reactions, which modify the common benz[a]anthraquinone carbon skeleton. In particular, the opposite stereochemistry of landomycins and urdamycins/gaudimycins at C-6 is generated by the short-chain alcohol dehydrogenases/reductases LanV and UrdMred/CabV, respectively. Here we present crystal structures of LanV and UrdMred in complex with NADP(+) and the product analog rabelomycin, which enabled us to identify four regions associated with the functional differentiation. The structural analysis was confirmed in chimeragenesis experiments focusing on these regions adjacent to the active site cavity, which led to reversal of the activities of LanV and CabV. The results surprisingly indicated that the conformation of the substrate and the stereochemical outcome of 6-ketoreduction appear to be intimately linked.
KeywordMeSH Terms
Protein Engineering
36. Kudo  F, Hoshi  S, Kawashima  T, Kamachi  T, Eguchi  T,     ( 2014 )

Characterization of a radical S-adenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis.

Journal of the American Chemical Society 136 (39)
PMID : 25230155  :   DOI  :   10.1021/ja507759f    
Abstract >>
The last step of neomycin biosynthesis is the epimerization at C-5? of neomycin C to give neomycin B. A candidate enzyme responsible for the epimerization was a putative radical S-adenosyl-L-methionine (SAM) enzyme, NeoN, which is uniquely encoded in the neomycin biosynthetic gene cluster and remained an unassigned protein in the neomycin biosynthesis. The reconstituted and reduced NeoN showed the expected epimerization activity in the presence of SAM. In the epimerization, 1 equiv of SAM was consumed to convert neomycin C into neomycin B. The site of neomycin C reactive toward epimerization was clearly confirmed to be C-5? by detecting the incorporation of a deuterium atom from the deuterium oxide-based buffer solution. Further, alanine scanning of the NeoN cysteine residues revealed that C249 is a critical amino acid residue that provides a hydrogen atom to complete the epimerization. Furthermore, electron paramagnetic resonance analysis of the C249A variant in the presence of SAM and neomycin C revealed that a radical intermediate is generated at the C-5? of neomycin C. Therefore, the present study clearly illustrates that the epimerization of neomycin C to neomycin B is catalyzed by a unique radical SAM epimerase NeoN with a radical reaction mechanism.
KeywordMeSH Terms
37. Khaliq  S, Ghauri  MA, Akhtar  K,     ( 2014 )

Characterization of mutations in regulatory genes of Tyl cluster leading to overexpression of tylosin in mutant �^-1 of Streptomyces fradiae NRRL-2702.

Applied microbiology and biotechnology 98 (2)
PMID : 24270892  :   DOI  :   10.1007/s00253-013-5317-8    
Abstract >>
Tylosin is a veterinary antibiotic and is commercially produced using Streptomyces fradiae. Previously, we developed a mutant �^-1 of S. fradiae NRRL-2702 with a 6.87-fold increase in tylosin yield as compared with the wild-type strain through irradiation mutagenesis. The present studies were conducted to explore mutational changes in regulatory genes (TylQ, TylP, TylS, TylR, and TylT) of Tyl cluster that may lead to an enhanced expression of tylosin. Expression analysis by RT-PCR revealed that TylQ was switched off earlier in mutant �^-1 while no change in expression pattern of TylP was observed between the wild-type and mutant �^-1 strains. However, a point mutation with a substitution of T to A was recorded at position 214 in the 420-bp product of TylP from mutant �^-1 that resulted in a change of one amino acid (serine to threonine) at position 72. Moreover, no mutation in the nucleotide sequence of TylS, TylR, and TylT genes was detected.
KeywordMeSH Terms
Genes, Regulator
Mutation
38. Forsman  M, Häggström  B, Lindgren  L, Jaurin  B,     ( 1990 )

Molecular analysis of beta-lactamases from four species of Streptomyces: comparison of amino acid sequences with those of other beta-lactamases.

Journal of general microbiology 136 (3)
PMID : 2391494  :   DOI  :   10.1099/00221287-136-3-589    
Abstract >>
Genes encoding extracellular beta-lactamases of Streptomyces badius, Streptomyces cacaoi, Streptomyces fradiae and Streptomyces lavendulae were cloned and mapped in Streptomyces lividans. DNA sequence analysis of the beta-lactamase genes revealed a high overall G + C content, ranging from 71 to 75 mol%, with a G + C content of 95 mol% at the third position of the codons for all four genes. The primary structure of the beta-lactamases including their signal peptides was deduced. The four beta-lactamases exhibited homology to each other and to class A beta-lactamases from other bacterial genera. We suggest that Streptomyces beta-lactamases are representatives of a superfamily of genes, from which class A beta-lactamases of Gram-negative bacteria may have evolved.
KeywordMeSH Terms
39. Metsä-Ketelä  M, Niemi  J, Rohr  J, Yang  K, Mäntsälä  P, Shaaban  KA, Klika  KD,     ( 2012 )

Tailoring enzymes involved in the biosynthesis of angucyclines contain latent context-dependent catalytic activities.

Chemistry & biology 19 (5)
PMID : 22633416  :   DOI  :   10.1016/j.chembiol.2012.04.010     PMC  :   PMC3361699    
Abstract >>
Comparison of homologous angucycline modification enzymes from five closely related Streptomyces pathways (pga, cab, jad, urd, lan) allowed us to deduce the biosynthetic steps responsible for the three alternative outcomes: gaudimycin C, dehydrorabelomycin, and 11-deoxylandomycinone. The C-12b-hydroxylated urdamycin and gaudimycin metabolites appear to be the ancestral representatives from which landomycins and jadomysins have evolved as a result of functional divergence of the ketoreductase LanV and hydroxylase JadH, respectively. Specifically, LanV has acquired affinity for an earlier biosynthetic intermediate resulting in a switch in biosynthetic order and lack of hydroxyls at C-4a and C-12b, whereas in JadH, C-4a/C-12b dehydration has evolved into an independent secondary function replacing C-12b hydroxylation. Importantly, the study reveals that many of the modification enzymes carry several alternative, hidden, or ancestral catalytic functions, which are strictly dependent on the biosynthetic context.
KeywordMeSH Terms
40. Siemieniak  DR, Slightom  JL, Chung  ST,     ( 1990 )

Nucleotide sequence of Streptomyces fradiae transposable element Tn4556: a class-II transposon related to Tn3.

Gene 86 (1)
PMID : 2155856  :   DOI  :   10.1016/0378-1119(90)90107-3    
Abstract >>
The first transposable element to be isolated from Streptomyces fradiae, Tn4556, was completely sequenced; the total of 6625 bp have an overall G + C composition of 68%. Computer-aided analysis of this sequence reveals the location of nine open reading frames (ORFs). Several of these ORFs, numbers 1, 2, and 7, contain ribosome-binding sites (RBS) near their putative translation-initiation sites, which share identity with the consensus RBS sequences of Escherichia coli and Bacillus subtilis. ORF1 potentially encodes an 892-amino acid (aa) protein and this deduced aa sequence shares 61% identity with that of the transposase encoded by the tnpA gene of Tn3. Three other ORFs, 2, 3 and 5, potentially encode proteins which are similar in size to the resolvase protein encoded by the Tn3 gene tnpR; however, none of the protein products deduced from these ORF share extensive aa sequence identity with other resolvase proteins.
KeywordMeSH Terms
DNA Transposable Elements
41. Clausnitzer  D, Piepersberg  W, Wehmeier  UF,     ( 2011 )

The oxidoreductases LivQ and NeoQ are responsible for the different 6'-modifications in the aminoglycosides lividomycin and neomycin.

Journal of applied microbiology 111 (3)
PMID : 21689223  :   DOI  :   10.1111/j.1365-2672.2011.05082.x    
Abstract >>
The 2-deoxystreptamine-containing aminoglycoside antibiotics (AGAs) constitute the largest subgroup of the aminoglycosides. Neomycin (NEO) and lividomycin (LIV) are both representatives of the pseudo-tetrasaccharide group among the NEO-type AGAs. While NEO contains a 6'-NH(2) group, the 6'-position remains unmodified in LIV. The aim of the study was to characterize the substrate specificities of the enzymes involved in the C-6'- and C-6?-modification in order to explain the different amination patterns. We overproduced and purified the enzymes NeoQ (bifunctional 6'- and 6?-oxidoreductase) and NeoB (bifunctional 6'- and-6?-aminotransferase), which had been analysed before (Huang et al. 2007), and compared the enzymatic properties with the corresponding enzymes LivQ (postulated 6?-oxidoreductase, 72% identity to NeoQ) and LivB (postulated 6?-aminotransferase, 71% identity to NeoB) from the LIV pathway. By applying a newly established photometric assay, we proved that LivQ oxidized only pseudotetrasaccharidic substrates at the 6?-position. In contrast, NeoQ accepted also the pseudodisaccharidic paromamine as a substrate and oxidized the 6'- and 6?-positions on two different precursors of NEO. The aminotransferases LivB and NeoB both transfer NH(2) groups to the 6'-position in the precursor 6'-oxo-paromamine and to the 6?-position of 6?-oxo-neomycin C. The difference in the modification pattern of NEO and LIV at their 6'-positions is based only on the difference in the substrate specificities of the oxidoreductases LivQ and NeoQ, respectively. The aminotransferases LivB and NeoB share identical biochemical properties, and both are capable to transaminate the 6' and also the 6?-position of the tested AGAs. Our data provide information to understand the structural variations in aminoglycosides and may be helpful to interpret variations in other natural product bisoynthesis pathways.
KeywordMeSH Terms
42.     ( 1996 )

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae.

Gene 180 (1��2��)
PMID : 8973363  :   DOI  :   10.1016/s0378-1119(96)00448-9    
Abstract >>
The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the TlrA and TlrD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related ('erm-type') genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.
KeywordMeSH Terms
43.     ( 1996 )

A general approach for cloning and characterizing dNDP-glucose dehydratase genes from actinomycetes.

FEMS microbiology letters 141 (2��3��)
PMID : 8768522  :   DOI  :   10.1111/j.1574-6968.1996.tb08384.x    
Abstract >>
Oligonucleotide primers were designed and successfully applied to amplify DNA fragments of dNDP-glucose dehydratase genes from actinomycete species producing natural compounds which contain deoxysugar moieties. The deduced amino acid sequence of the isolated fragments revealed similarity to known dNDP-glucose dehydratases. A phylogeny for the deduced proteins of the obtained fragments and for dNDP-glucose dehydratases described in the data bases was constructed. dNDP-glucose dehydratases from actinomycetes were more related to each other than to dehydratases from species of other orders. The phylogenetic analysis also revealed a close relation between dehydratases from strains producing natural compounds with similar deoxysugar moieties.
KeywordMeSH Terms
Cloning, Molecular
44.     ( 1997 )

Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome.

Gene 184 (2)
PMID : 9031628  :   DOI  :   10.1016/s0378-1119(96)00595-1    
Abstract >>
The tylLM region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains four open reading frames (orfs1*-4*). The function of the orf1* product is not known. The product of orf2* (tylM2) is the glycosyltransferase that adds mycaminose to the 5-hydroxyl group of tylactone, the polyketide aglycone of tylosin (Ty). A methyltransferase, responsible for 3-N-methylation during mycaminose production, is encoded by orf3* (tylM1). The product of orf4* (cer) is crotonyl-CoA reductase, which converts acetoacetyl-CoA to butyryl-CoA for use as a 4C extender unit during tylactone production.
KeywordMeSH Terms
45.     ( 1996 )

New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces.

Biochimica et biophysica acta 1292 (2)
PMID : 8597568  :   DOI  :   10.1016/0167-4838(95)00207-3    
Abstract >>
Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.
KeywordMeSH Terms
46.     ( 1993 )

Purification, characterization and molecular cloning of an acidic amino acid-specific proteinase from Streptomyces fradiae ATCC 14544.

Biochimica et biophysica acta 1163 (2)
PMID : 8490047  :   DOI  :   10.1016/0167-4838(93)90176-r    
Abstract >>
We have isolated a novel acidic amino-acid-specific proteinase from Streptomyces fradiae ATCC 14544, using benzyloxycarbonyl-L-Phe-L-Leu-L-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. A proteinase, which we propose to call SFase, was purified from the culture filtrate by salting out, repeated S-Sepharose chromatography, and affinity chromatography (CH-Sepharose-Phe-Leu-D-Glu-OMe). The purified enzyme showed a single band having an apparent molecular weight of 19,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When synthetic peptides were used as substrates, SFase showed high specificity for Z-Phe-Leu-Glu-pNA. Comparison with nitroanilides of glutamic acid and aspartic acid as substrates revealed that the reactivity was about 10-fold higher for a glutamyl bond than an aspartyl bond. SFase selectively hydrolyzed the -Glu-Ala-bond of two glutamyl bonds in the oxidized insulin B-chain within the initial reaction time until the starting material was completely digested. Diisopropylfluorophosphate and benzyloxycarbonyl-Phe-Leu-Glu chloromethylketone completely inhibited SFase, while metalloproteinase inhibitors, such as EDTA and o-phenanthrolin, did not inhibit the enzyme. The findings indicate that SFase can be classified as a serine proteinase, and is highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure and precursor of SFase, its gene was cloned from genomic DNA of the producing strain, and the nucleotide sequence was determined. Consideration of the N- and C-terminal amino-acid sequences of the mature protein of SFase indicates that it consists of 187 amino acids, which follows a prepropeptide of 170 residues. In comparison with the acidic amino-acid-specific proteinase from Streptomyces griseus (Svendsen, I., Jensen, M.R. and Breddam, K. (1991) FEBS Lett. 292, 165-167), SFase had 82% homology in the amino acid sequence. The processing site for maturation of SFase was a unique sequence (-Glu-Val-), so that the propeptide could be released by cleavage of the peptide bond between Glu and Val.
KeywordMeSH Terms
47.     ( 1994 )

Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544.

European journal of biochemistry 220 (1)
PMID : 8119298  :   DOI  :   10.1111/j.1432-1033.1994.tb18598.x    
Abstract >>
A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase. The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase-2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1-6]. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.
KeywordMeSH Terms
48.     ( 1994 )

Sequence around the centromere of Saccharomyces cerevisiae chromosome II: similarity of CEN2 to CEN4.

Yeast (Chichester, England) 10 Suppl A (N/A)
PMID : 8091860  :   DOI  :   10.1002/yea.320100006    
Abstract >>
We report the sequence of a 12 kilobase region spanning the centromere of Saccharomyces cerevisiae chromosome II. The sequence from the left arm includes genes for histones H2A and H2B. The sequence from the right arm includes a gene that probably encodes a novel trehalase, as well as the COQ1 gene (for an enzyme involved in coenzyme Q biosynthesis), and an open reading frame with significant similarity to bacterial genes of unknown function. The trehalase gene (YBR0106) on chromosome II is located beside the centromere and transcribed towards it. This is identical to the arrangement of the neutral trehalase gene (NTH1) beside the centromere of chromosome IV. The centromere regions of chromosomes II and IV may therefore have arisen through a duplication of the centromere region of an ancestral chromosome. The YBR0106 and NTH1 proteins are 77% identical in predicted amino acid sequence, but there is no pronounced sequence similarity between the two centromeres (CEN2 and CEN4) outside of the universally conserved CDE I and CDE III elements. The genes flanking the centromere and trehalase genes differ between the two chromosomes, so the similarity between chromosomes II and IV may be less extensive than that recently reported between chromosomes III and XIV.
KeywordMeSH Terms
Alkyl and Aryl Transferases
Centromere
Chromosomes, Fungal
Genes, Fungal
49.     ( 1994 )

Analysis of five tylosin biosynthetic genes from the tyllBA region of the Streptomyces fradiae genome.

Molecular microbiology 13 (2)
PMID : 7984112  :   DOI  :   10.1111/j.1365-2958.1994.tb00428.x    
Abstract >>
The tyllBA region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains at least five open reading frames (ORFs). ORF1 (tylI) encodes a cytochrome P450 and mutations in this gene affect macrolide ring hydroxylation. The product of ORF2 (tylB) belongs to a widespread family of proteins whose functions are speculative, although tylB mutants are defective in the biosynthesis or addition of mycaminose during tylosin production. ORFs 3 and 4 (tylA1 and tylA2) encode delta TDP-glucose synthase and delta TDP-glucose dehydratase, respectively, enzymes responsible for the first two steps common to the biosynthesis of all three deoxyhexose sugars of tylosin via the common intermediate, delta TDP-4-keto, 6-deoxyglucose. ORF5 encodes a thioesterase similar to one encoded in the erythromycin gene cluster of Saccharopolyspora erythraea.
KeywordMeSH Terms
Genes, Bacterial
Mannose-6-Phosphate Isomerase
50.     ( 1994 )

Crystal structure analysis of a serine proteinase from Streptomyces fradiae at 0.16-nm resolution and molecular modeling of an acidic-amino-acid-specific proteinase.

European journal of biochemistry 224 (2)
PMID : 7925392  :   DOI  :   10.1111/j.1432-1033.1994.00735.x    
Abstract >>
We have determined the three-dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase-2) at 0.16-nm resolution. SFase-2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur. J. Biochem. 220, 55-61]. In the present study, data were collected to approximately 0.16-nm resolution on a Rigaku R-AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined structure of Streptomyces griseus protease A [Sielecki, A. R., Hendrickson, W. A., Broughton, C. G., Delbaere, L. T., Brayer, G. D. & James, M. N. (1979) J. Mol. Biol. 134, 781-804]. The starting model gave an initial crystallographic R factor of 0.443. Refinement with restrained least-squares converged at a final R factor of 0.182 for 16128 observed reflections. The final model includes 86 water molecules. The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a beta barrel with six-stranded beta sheets and two alpha helices. The overall tertiary structure of SFase-2 is similar to the structures of other chymotrypsin-like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar three-dimensional structures, indicating that a gene duplication event has occurred to produce these two domains. We predicted the tertiary structure of an acidic-amino-acid-specific proteinase on the basis of the crystal structure of SFase-2, and compared the active-site conformations of these two enzymes. We found a characteristic histidine cluster of three histidine residues in the active site of the acidic-amino-acid-specific proteinase. The substrate recognition mechanism of SFase-2 may be mediated through the hydrophobic amino acid residues. However, in the acidic-amino-acid-specific proteinase, the positive charge of this histidine cluster would attract the negative charges of glutamic acid and aspartic acid.
KeywordMeSH Terms
Models, Molecular
Protein Structure, Secondary
51. Decker  H, Haag  S,     ( 1995 )

Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae T?2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation.

Journal of bacteriology 177 (21)
PMID : 7592377  :   DOI  :   10.1128/jb.177.21.6126-6136.1995     PMC  :   PMC177452    
Abstract >>
A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae T?2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C.
KeywordMeSH Terms
Genes, Bacterial
52.     ( 1994 )

Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae.

Journal of bacteriology 176 (19)
PMID : 7928973  :   DOI  :   10.1128/jb.176.19.6107-6119.1994     PMC  :   PMC196831    
Abstract >>
Targeted inactivation of the valine (branched-chain amino acid) dehydrogenase gene (vdh) was used to study the role of valine catabolism in the production of tylosin in Streptomyces fradiae and spiramycin in Streptomyces ambofaciens. The deduced products of the vdh genes, cloned and sequenced from S. fradiae C373.1 and S. ambofaciens ATCC 15154, are approximately 80% identical over all 363 amino acids and 96% identical over a span of the first N-terminal 107 amino acids, respectively, to the deduced product of the Streptomyces coelicolor vdh gene. The organization of the regions flanking the vdh genes is the same in all three species. Inactivation of the genomic copy of the vdh gene in S. fradiae and S. ambofaciens by insertion of a hygromycin resistance (hyg) gene caused loss of the valine dehydrogenase (Vdh) activity, and thus only one enzyme is responsible for the Vdh activity in these organisms. Analysis of the culture broth by bioassay revealed that the vdh::hyg mutants produce an approximately sixfold-lower level of tylosin and an approximately fourfold-lower level of spiramycin than the wild-type S. fradiae and S. ambofaciens strains, while maintaining essentially identical growth in a defined minimal medium with either 25 mM ammonium ion or 0.05% asparagine as the nitrogen source. The addition of the valine catabolite, propionate or isobutyrate, and introduction of the wild-type vdh gene back to each vdh::hyg mutant reversed the negative effect of the vdh::hyg mutation on spiramycin and tylosin production. These data show that the catabolism of valine is a major source of fatty acid precursors for macrolide biosynthesis under defined growth conditions and imply that amino acid catabolism is a vital source of certain antibiotic precursors in actinomycetes.
KeywordMeSH Terms
Cinnamates
53. Taguchi  S, Kikuchi  H, Kojima  S, Kumagai  I, Nakase  T, Miura  K, Momose  H,     ( 1993 )

High frequency of SSI-like protease inhibitors among Streptomyces.

Bioscience, biotechnology, and biochemistry 57 (3)
PMID : 7763545  :  
Abstract >>
N/A
KeywordMeSH Terms
54. Kurai  S, Urabe  H, Ogawara  H,     ( 1995 )

Cloning, sequencing, and site-directed mutagenesis of beta-lactamase gene from Streptomyces fradiae Y59.

Antimicrobial agents and chemotherapy 39 (1)
PMID : 7535038  :   DOI  :   10.1128/aac.39.1.260     PMC  :   PMC162522    
Abstract >>
The beta-lactamase gene from Streptomyces fradiae Y59 was cloned and sequenced. To determine which amino acid residues are critical in binding activity to blue dextran, chimera beta-lactamases were constructed and their binding abilities were determined. The results suggested that blue dextran binding may depend more on overall conformation of about two-thirds of the beta-lactamase molecule from the N terminus than on the primary structure.
KeywordMeSH Terms
55. Seno  ET, Baltz  RH,     ( 1981 )

Properties of S-adenosyl-L-methionine:macrocin O-methyltransferase in extracts of Streptomyces fradiae strains which produce normal or elevated levels of tylosin and in mutants blocked in specific O-methylations.

Antimicrobial agents and chemotherapy 20 (3)
PMID : 7305323  :   DOI  :   10.1128/aac.20.3.370     PMC  :   PMC181703    
Abstract >>
An efficient assay for S-adenosyl-L-methionine:macrocin O-methyltransferase, the enzyme which carries out the terminal step in tylosin biosynthesis, is described. Macrocin O-methyltransferase requires Mg2+ and S-adenosyl-L-methionine for activity, has a temperature optimum of about 31 degrees C, and has a pH optimum of 7.5 to 8.2. Macrocin O-methyltransferase specifically converts macrocin to tylosin by O-methylation of the 3" ' position of macrocin. In vitro methylation studies with extracts from a tylosin-producing Streptomyces fradiae strain and from mutant strains blocked in 2" '- or 3" '-O-methylations indicated that: (i) the 2" '- and 3" '-O-methylations occur after 6-deoxy-D-allose is attached to the macrolide ring; (ii) the 2" '- and 3" '-O-methylations are carried out by separate enzymes; and (iii) the 2" '-O-methylation precedes the 3" '-O-methylation. Macrocin O-methyltransferase was inhibited by high levels of its substrate, macrocin, by its product, tylosin, and by other tylosin analogs which contained mycinose or demethyl analogs of mycinose. Macrocin O-methyltransferase was produced early in the tylosin fermentation cycle by S. fradiae and preceded the onset of rapid tylosin biosynthesis by about 24 h. The enzyme specific activity reached maximum at about 72 h and then slowly declined. A mutant strain of S. fradiae selected for increased tylosin production synthesized macrocin O-methyltransferase more rapidly and accumulated a higher enzyme specific activity than a wild-type strain.
KeywordMeSH Terms
56. Thompson  CJ, Gray  GS,     ( 1983 )

Nucleotide sequence of a streptomycete aminoglycoside phosphotransferase gene and its relationship to phosphotransferases encoded by resistance plasmids.

Proceedings of the National Academy of Sciences of the United States of America 80 (17)
PMID : 6310563  :   DOI  :   10.1073/pnas.80.17.5190     PMC  :   PMC384217    
Abstract >>
The DNA sequence of an aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae ATCC 10745 (a neomycin producer) was determined. The gene was localized by in vitro subcloning and insertional inactivation. Molecular weight, amino acid composition, and amino-terminal analysis of the purified aph gene product confirmed the accuracy and position of the aph gene sequence. Pairwise comparisons of S. fradiae aph with the aph genes encoded by bacterial transposons Tn5 and Tn903 showed significant nucleotide and amino acid homologies which indicated a common evolutionary origin for these antibiotic-resistance genes.
KeywordMeSH Terms
Genes
Genes, Bacterial
Plasmids
57. Baltz  RH, Seno  ET,     ( 1981 )

Properties of Streptomyces fradiae mutants blocked in biosynthesis of the macrolide antibiotic tylosin.

Antimicrobial agents and chemotherapy 20 (2)
PMID : 7283418  :   DOI  :   10.1128/aac.20.2.214     PMC  :   PMC181666    
Abstract >>
We isolated numerous mutants of Streptomyces fradiae blocked in tylosin biosynthesis after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were classified into nine groups, based upon the tylosin-like compounds produced and upon cofermentation analyses. More than 80% of the mutants isolated produced no tylosin-like compounds, and the majority of these were blocked only in the formation of tylactone. Four classes of mutants blocked in the biosynthesis or addition of tylosin sugars were isolated; tylA mutants were blocked in the formation of all three tylosin sugars, whereas tylB, tylC, and tylD mutants were blocked specifically in the biosynthesis or the addition of mycaminose, mycarose, and 6-deoxy-d-allose, respectively. Two classes of mutants (tylH and tylI) blocked in specific oxidations of tylactone and two classes (tylE and tylF) blocked in specific O-methylations of demethylmacrocin and macrocin were also characterized. Cofermentation and bioconversion studies with these mutants suggested the following relationships: (i) the tylosin sugars are derived from a common intermediate; (ii) tylactone is the first intermediate which can be excreted in appreciable quantities; (iii) the addition of mycaminose to the C-5 hydroxyl group of tylactone must precede oxidations at C-20 and C-23; (iv) oxidation at C-20 normally precedes the attachment of mycarose to the 4' hydroxyl position of mycaminose; and (v) 6-deoxy-d-allose is added to the C-23 hydroxyl position of the lactone and subsequently O-methylated at 2''' and 3''' positions. The O-methylations appear to be the final two steps in tylosin biosynthesis, and the 2''' O-methylation must occur before the 3''' O-methylation can take place. All of the tyl mutants except the tylG mutants produced relatively high levels of tylosin-like intermediates or shunt products. Mutants blocked in specific steps other than 3''' O-methylation, including a mutant blocked in 2''' O-methylation of demethylmacrocin, produced normal levels of macrocin O-methyltransferase. Mutants apparently containing specific tylosin structural gene mutations produced normal levels of aerial mycelia and spores, produced low levels of tylosin aldehyde reductase, and were resistant to high levels of tylosin. However, three atypical tylG mutants produced no tylosin-like compounds, could not cosynthesize tylosin with any other tyl mutant, could not bioconvert tylactone or macrocin to tylosin, and produced no macrocin O-methyltransferase. These three mutants produced elevated levels of tylosin aldehyde reductase. In addition, one was very succeptible to tylosin and did not produce aerial mycelia or spores.
KeywordMeSH Terms
58. Bauer  NJ, Kreuzman  AJ, Dotzlaf  JE, Yeh  WK,     ( 1988 )

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine:macrocin O-methyltransferase from Streptomyces fradiae.

The Journal of biological chemistry 263 (30)
PMID : 3170601  :  
Abstract >>
S-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required Mg2+, Mn2+, or Co2+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 degrees C. The O-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:1. The enzyme also mediated conversion of lactenocin----desmycosin. The corresponding Vmax/Km ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin O-methyltransferase.
KeywordMeSH Terms
59. Kamimiya  S, Weisblum  B,     ( 1988 )

Translational attenuation control of ermSF, an inducible resistance determinant encoding rRNA N-methyltransferase from Streptomyces fradiae.

Journal of bacteriology 170 (4)
PMID : 3127381  :   DOI  :   10.1128/jb.170.4.1800-1811.1988     PMC  :   PMC211034    
Abstract >>
An inducible resistance determinant, ermSF, from the tylosin producer Streptomyces fradiae NRRL 2338 has been cloned, sequenced, and shown to confer inducible macrolide-lincosamide-streptogramin B resistance when transferred to Streptomyces griseofuscus NRRL 23916. From mapping studies with S1 nuclease to locate the site of transcription initiation, the ermSF message contains a 385-nucleotide 5' leader sequence upstream from the 960-nucleotide major open reading frame that encodes the resistance determinant. On the basis of the potential secondary structure that the ermSF leader can assume, a translational attenuation model similar to that for ermC is proposed. The model is supported by mutational analysis involving deletions in the proposed attenuator. By analysis with restriction endonucleases, ermSF is indistinguishable from the tlrA gene described by Birmingham et al. (V. A. Birmingham, K. L. Cox, J. L. Larson, S. E. Fishman, C. L. Hershberger, and E. T. Seno, Mol. Gen. Genet. 204:532-539, 1986) which comprises one of at least three genes from S. fradiae that can confer tylosin resistance when subcloned into S. griseofuscus. When tested for inducibility, ermSF appears to be strongly induced by erythromycin, but not by tylosin.
KeywordMeSH Terms
Gene Expression Regulation
Macrolides
Protein Biosynthesis
60. Fishman  SE, Cox  K, Larson  JL, Reynolds  PA, Seno  ET, Yeh  WK, Van Frank  R, Hershberger  CL,     ( 1987 )

Cloning genes for the biosynthesis of a macrolide antibiotic.

Proceedings of the National Academy of Sciences of the United States of America 84 (23)
PMID : 3479787  :   DOI  :   10.1073/pnas.84.23.8248     PMC  :   PMC299519    
Abstract >>
Macrocin-O-methyltransferase (MacOMeTase) catalyzes the final enzymatic step in the biosynthesis of tylosin in Streptomyces fradiae. A 44-base mixed oligonucleotide probe containing only guanosine and cytidine in the third position of degenerate codons was synthesized based on the amino acid sequence of the amino terminus of MacOMeTase. Plaque blot hybridization to a bacteriophage lambda library and colony blot hybridization to a cosmid library of S. fradiae DNA identified recombinants that contained overlapping fragments of chromosomal DNA. The nucleotide sequence of the cloned DNA verified that the DNA contained the coding sequence for MacOMeTase. Recombinant plasmids transformed mutants blocked in tylosin biosynthesis and complemented tylF (the structural gene for MacOMeTase) and tyl mutations of eight other classes.
KeywordMeSH Terms
61. Kreuzman  AJ, Turner  JR, Yeh  WK,     ( 1988 )

Two distinctive O-methyltransferases catalyzing penultimate and terminal reactions of macrolide antibiotic (tylosin) biosynthesis. Substrate specificity, enzyme inhibition, and kinetic mechanism.

The Journal of biological chemistry 263 (30)
PMID : 3170602  :  
Abstract >>
S-Adenosyl-L-methionine:demethylmacrocin O-methyltransferase catalyzes the conversion of demethylmacrocin to macrocin as the penultimate step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was purified to electrophoretic homogeneity by a conventional chromatographic procedure. The purified enzyme appears to be trimeric with a molecular weight of 122,000-126,000 and a subunit size of 42,000. Its isoelectric point was 6.0. The enzyme required Mg2+ for maximal activity and was catalytically optimal at pH 7.8-8.5 and 42 degrees C. The O-methyltransferase catalyzed conversion of demethylmacrocin to macrocin at a stoichiometric ratio of 1:1. The O-methyltransferase also mediated conversion of demethyllactenocin----lactenocin. The corresponding Vmax/Km ratios for the two analogous conversions varied only slightly. Both enzymic conversions were susceptible to an extensive and identical range of metabolic inhibitions. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions are consistent with Ordered Bi Bi as the reaction mechanism of demethylmacrocin O-methyltransferase. Except for an identical kinetic mechanism, demethylmacrocin O-methyltransferase can be readily differentiated from macrocin O-methyltransferase by its physical and catalytic properties as well as metabolic inhibitions.
KeywordMeSH Terms
62. Sato  S, Miyanaga  A, Kim  SY, Kuzuyama  T, Kudo  F, Eguchi  T,     ( 2018 )

Biochemical and Structural Analysis of FomD That Catalyzes the Hydrolysis of Cytidylyl (S)-2-Hydroxypropylphosphonate in Fosfomycin Biosynthesis.

Biochemistry 57 (32)
PMID : 30010320  :   DOI  :   10.1021/acs.biochem.8b00690    
Abstract >>
In fosfomycin biosynthesis, the hydrolysis of cytidylyl (S)-2-hydroxypropylphosphonate [(S)-HPP-CMP] to afford (S)-HPP is the only uncharacterized step. Because FomD is an uncharacterized protein with a DUF402 domain that is encoded in the fosfomycin biosynthetic gene cluster, FomD was hypothesized to be responsible for this reaction. In this study, FomD was found to hydrolyze (S)-HPP-CMP to give (S)-HPP and CMP efficiently in the presence of Mn2+ or Co2+. FomD also hydrolyzed cytidylyl 2-hydroxyethylphosphonate (HEP-CMP), which is a biosynthetic intermediate before C-methylation. The kcat/ KM value of FomD with (S)-HPP-CMP was 10-fold greater than that with HEP-CMP, suggesting that FomD hydrolyzes (S)-HPP-CMP rather than HEP-CMP in bacteria. The crystal structure of FomD showed that this protein adopts a barrel-like fold, which consists of a large twisted antiparallel �]-sheet. This is a key structural feature of the DUF402 domain-containing proteins. Two metal cations are located between the FomD barrel and the two �\-helices at the C-terminus and serve to presumably activate the phosphonate group of substrates for hydrolysis. Docking simulations with (S)-HPP-CMP suggested that the methyl group at the C2 position of the HPP moiety is recognized by a hydrophobic interaction with Trp68. Further mutational analysis suggested that a conserved Tyr107 among the DUF402 domain family of proteins activates a water molecule to promote nucleophilic attack on the phosphorus atom of the phosphonate moiety. These findings provide mechanistic insights into the FomD reaction and lead to a complete understanding of the fosfomycin biosynthetic pathway in Streptomyces.
KeywordMeSH Terms
63. Bibb  MJ, Bibb  MJ, Ward  JM, Cohen  SN,     ( 1985 )

Nucleotide sequences encoding and promoting expression of three antibiotic resistance genes indigenous to Streptomyces.

Molecular & general genetics : MGG 199 (1)
PMID : 2987648  :   DOI  :   10.1007/bf00327505    
Abstract >>
Promoter-probe plasmid vectors were used to isolate putative promoter-containing DNA fragments of three Streptomyces antibiotic resistance genes, the rRNA methylase (tsr) gene of S. azureus, the aminoglycoside phosphotransferase (aph) gene of S. fradiae, and the viomycin phosphotransferase (vph) gene of S. vinaceus. DNA sequence analysis was carried out for all three of the fragments and for the protein-coding regions of the tsr and vph genes. No sequences resembling typical E. coli promoters or Bacillus vegetatively-expressed promoters were identified. Furthermore, none of the three DNA fragments found to be transcriptionally active in Streptomyces could initiate transcription when introduced into E. coli. An extremely biased codon usage pattern that reflects the high G + C composition of Streptomyces DNA was observed for the protein-coding regions of the tsr and vph genes, and of the previously sequenced aph gene. This pattern enabled delineation of the protein-coding region and identification of the coding strand of the genes.
KeywordMeSH Terms
Drug Resistance, Microbial
Gene Expression Regulation
64. Ray  S, Maitra  A, Biswas  A, Panjikar  S, Mondal  J, Anand  R,     ( 2017 )

Functional insights into the mode of DNA and ligand binding of the TetR family regulator TylP from Streptomyces fradiae.

The Journal of biological chemistry 292 (37)
PMID : 28739805  :   DOI  :   10.1074/jbc.M117.788000     PMC  :   PMC5602390    
Abstract >>
Tetracycline repressors (TetRs) modulate multidrug efflux pathways in several pathogenic bacteria. In Streptomyces, they additionally regulate secondary metabolic pathways like antibiotic production. For instance, in the antibiotic producer Streptomyces fradiae, a layered network of TetRs regulates the levels of the commercially important antibiotic tylosin, with TylP occupying the top of this cascading network. TetRs exist in two functional states, the DNA-bound and the ligand-bound form, which are allosterically regulated. Here, to develop deeper insights into the factors that govern allostery, the crystal structure of TylP was solved to a resolution of 2.3 ?. The structure revealed that TylP possesses several unique features; notably, it harbors a unique C-terminal helix-loop extension that spans the entire length of the structure. This anchor connects the DNA-binding domain (DBD) with the ligand-binding domain (LBD) via a mix of positively charged and hydrogen-bonding interactions. Supporting EMSA studies with a series of �GC truncated versions show that a systematic deletion of this region results in complete loss of DNA binding. The structure additionally revealed that TylP is markedly different in the orientation of its DBD and LBD architecture and the dimeric geometry from its hypothesized Streptomyces homologue CprB, which is a �^-butyrolactone regulator. Rather, TylP is closer in structural design to macrolide-binding TetRs found in pathogens. Supporting molecular dynamic studies suggested that TylP binds a macrolide intermediate in the tylosin pathway. Collectively, the structure along with corroborating biochemical studies provided insights into the novel mode of regulation of TetRs in antibiotic-producing organisms.
KeywordMeSH Terms
C-terminal extension
DNA binding
DNA-binding protein
TetR family regulator
TylP
X-ray crystallography
actinobacteria
bacterial metabolism
quorum sensing
tylosin regulation
C-terminal extension
DNA binding
DNA-binding protein
TetR family regulator
TylP
X-ray crystallography
actinobacteria
bacterial metabolism
quorum sensing
tylosin regulation
Models, Molecular
65.     ( 1998 )

Characterization and targeted disruption of a glycosyltransferase gene in the tylosin producer, Streptomyces fradiae.

Gene 214 (1��2��)
PMID : 9651492  :   DOI  :   10.1016/s0378-1119(98)00210-8    
Abstract >>
An open reading frame, designated tylN, has been identified by sequence analysis at one end of the tylosin biosynthetic gene cluster of Streptomyces fradiae, alongside a cluster of genes encoding the biosynthesis of dTDP-deoxyallose. This 6-deoxyhexose sugar is converted to mycinose, via bis O-methylation, following attachment to the polyketide lactone during tylosin biosynthesis. The deduced product of tylN is similar to several glycosyltransferases, authentic and putative, and displays a consensus sequence motif that appears to be characteristic of a sub-group of such enzymes. Specific disruption of tylN within the S. fradiae genome resulted in the production of demycinosyl-tylosin, whereas other glycosyltransferase activities involved in tylosin biosynthesis were not affected. Evidently, tylN encodes deoxyallosyl transferase.
KeywordMeSH Terms
Genes, Bacterial

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