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Jae Kyung Sohng - One of the best experts on this subject based on the ideXlab platform.

  • implication of orphan histidine kinase ohkasp in biosynthesis of doxorubicin and daunorubicin in Streptomyces Peucetius atcc 27952
    Microbiological Research, 2018
    Co-Authors: Anaya Raj Pokhrel, Amit Kumar Chaudhary, Dipesh Dhakal, Hue Thi Nguyen, Jae Kyung Sohng
    Abstract:

    Abstract The orphan histidine kinase (HK) from Streptomyces Peucetius ATCC 27952 ( ohkAsp ) was found to be implicated in the regulation of doxorubicin (DOX)/daunorubicin (DNR) biosynthesis, self-defense and developmental attributes. OhkAsp is a homolog of OhkA from Streptomyces coelicolor and Streptomyces avermitilis (with 73 and 75% identity). As in its homologs, S. Peucetius mutant with deletion of ohkAsp was found to enhance metabolite biosynthesis and impaired the morphological differentiation. But, unlike its homologs from Streptomyces coelicolor and Streptomyces avermitilis, differential enhancement in level of secondary metabolite production was found in overexpression mutants apart from deletion mutant. The deflection in characteristics of OhkA in its homologue from S. Peucetius ATCC 27952, and its imminent implications was monitered by making various mutants with differential expression level of ohkAsp . The variations were observed in the morphology of mutants, transcriptional level of effectors and regulators of DOX/DNR biosynthesis pathway, DOX/DNR precursor pool and biomass accumulation. Based on comparisons of domain arrangements among its homologs, Low Complexity Region (LCR) present on the OhkAsp was the only domain that stood out. Further, the LCR on OhkAsp was found to be overlapping with a putative receiver domain responsible for interaction with response regulator. The imminent implications of differential expression level of ohkAsp on: regulation and biosynthesis of DOX/DNR, morphological differentiation, DOX/DNR precursor pool and biomass accumulation were explored in this study.

  • genome guided exploration of metabolic features of Streptomyces Peucetius atcc 27952 past current and prospect
    Applied Microbiology and Biotechnology, 2018
    Co-Authors: Nguyen Huy Thuan, Dipesh Dhakal, Anaya Raj Pokhrel, Thi Thuy Van Pham, Anil Shrestha, Jae Kyung Sohng
    Abstract:

    Streptomyces Peucetius ATCC 27952 produces two major anthracyclines, doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of several cancers. In order to gain detailed insight on genetics and biochemistry of the strain, the complete genome was determined and analyzed. The result showed that its complete sequence contains 7187 protein coding genes in a total of 8,023,114 bp, whereas 87% of the genome contributed to the protein coding region. The genomic sequence included 18 rRNA, 66 tRNAs, and 3 non-coding RNAs. In silico studies predicted ~ 68 biosynthetic gene clusters (BCGs) encoding diverse classes of secondary metabolites, including non-ribosomal polyketide synthase (NRPS), polyketide synthase (PKS I, II, and III), terpenes, and others. Detailed analysis of the genome sequence revealed versatile biocatalytic enzymes such as cytochrome P450 (CYP), electron transfer systems (ETS) genes, methyltransferase (MT), glycosyltransferase (GT). In addition, numerous functional genes (transporter gene, SOD, etc.) and regulatory genes (afsR-sp, metK-sp, etc.) involved in the regulation of secondary metabolites were found. This minireview summarizes the genome-based genome mining (GM) of diverse BCGs and genome exploration (GE) of versatile biocatalytic enzymes, and other enzymes involved in maintenance and regulation of metabolism of S. Peucetius. The detailed analysis of genome sequence provides critically important knowledge useful in the bioengineering of the strain or harboring catalytically efficient enzymes for biotechnological applications.

  • complete genome sequence of Streptomyces Peucetius atcc 27952 the producer of anticancer anthracyclines and diverse secondary metabolites
    Journal of Biotechnology, 2018
    Co-Authors: Dipesh Dhakal, Tokutaro Yamaguchi, Jae Kyung Sohng
    Abstract:

    Abstract Streptomyces Peucetius ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of S. Peucetius ATCC 27952, which consists of 8,023,114 bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed ∼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, in silico analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes.

  • substrate scope of o methyltransferase from Streptomyces Peucetius for biosynthesis of diverse natural products methoxides
    Applied Biochemistry and Biotechnology, 2017
    Co-Authors: Prakash Parajuli, Dipesh Dhakal, Ramesh Prasad Pandey, Thi Huyen Trang Nguyen, Jae Kyung Sohng
    Abstract:

    : Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces Peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. Peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.

  • overexpression of a pathway specific negative regulator enhances production of daunorubicin in blda deficient Streptomyces Peucetius atcc 27952
    Microbiological Research, 2016
    Co-Authors: Anaya Raj Pokhrel, Kwangkyoung Liou, Dipesh Dhakal, Amit Kumar Chaudhary, Anil Shrestha, Hue Thi Nguyen, Tuoi Thi Le, Jae Kyung Sohng
    Abstract:

    Abstract The dnrO gene is the first regulator to be activated in the daunorubicin (DNR) biosynthesis pathway of Streptomyces Peucetius ATCC 27952. DnrO is known for its self-repression capability while it activates rest of the DNR biosynthesis pathway through cascades of regulatory events. S. Peucetius was found to contain no functional copy of bldA-tRNA while a detailed examination of dnrO codons reveals the presence of TTA codon, which is rarely encoded by bldA-tRNA. Therefore, for evaluating the role of dnrO in DNR production, multiple engineered strains of S. Peucetius were generated by heterologously expressing bldA, dnrO and combination of bldA and dnrO. Using these strains, the effects of heterologously expressed bldA and overexpressed dnrO were evaluated on pathway specific regulators, mycelial densities and production of DNR. The results showed that the transcription level of dnrO and master regulator dnrI, was found to be elevated in bldA containing strain in comparison to dnrO overexpressed strain. The bldA containing strain produces 45.7% higher DNR than bldA deficient wild type strain from culture broth with OD600 of 1.45 at 72 h. Heterologous expression of bldA–tRNA is accounted for increased transcription levels of the DNR pathway specific regulators and enhanced DNR production.

Taejin Oh - One of the best experts on this subject based on the ideXlab platform.

  • crystal structure of cytochrome p450 cyp105p2 from Streptomyces Peucetius and its conformational changes in response to substrate binding
    International Journal of Molecular Sciences, 2016
    Co-Authors: Hemraj Rimal, Hyun Jin Park, Taejin Oh
    Abstract:

    Cytochrome P450 monooxygenases (CYP, EC 1.14.14.1) belong to a large family of enzymes that catalyze the hydroxylation of various substrates. Here, we present the crystal structure of CYP105P2 isolated from Streptomyces Peucetius ATCC27952 at a 2.1 A resolution. The structure shows the presence of a pseudo-ligand molecule in the active site, which was co-purified fortuitously and is presumed to be a biphenyl derivative. Comparison with previously determined substrate-bound CYP structures showed that binding of the ligand produces large and distinctive conformational changes in α2–α3, α7–α9, and the C-terminal loop regions. This structural flexibility confirms our previous observation that CYP105P2 can accommodate a broad range of ligands. The structure complexed with a pseudo-ligand provides the first molecular view of CYP105P2–ligand interactions, and it indicates the involvement of hydrophobic residues (Pro82, Ala181, Met187, Leu189, Leu193, and Ile236) in the interactions between hydrophobic ligands and CYP105P2. These results provide useful insights into the structural changes involved in the recognition of different ligands by CYP105P2.

  • understanding of real alternative redox partner of Streptomyces Peucetius doxa prediction and validation using in silico and in vitro analyses
    Archives of Biochemistry and Biophysics, 2015
    Co-Authors: Hemraj Rimal, Taejin Oh
    Abstract:

    Streptomyces Peucetius ATCC27952 contains the cytochrome P450 monoxygenase DoxA that is responsible for the hydroxylation of daunorubicin into doxorubicin. Although S. Peucetius ATCC27952 contains several potential redox partners, the most suitable endogenous electron-transport system is still unclear; therefore, we conducted a study of potential redox partners using Accelrys Discovery Studio 3.5. Recombinant DoxA along with its redox partners from S. Peucetius FDX1, FDR2, and FDX3, and the putidaredoxin and putidaredoxin reductase from Pseudomonas putida that are essential equivalents of the class I type of bacterial electron-transport system were over-expressed and purified. The successful development of an efficient redox system was achieved by an in vitro enzymatic catalysis reaction with DoxA. The optimal pH for the activation of the heme was 7.6 and the optimal temperature was 30 °C. Our findings suggest a two-fold increase of DoxA activity via the NADH → FDR2 → FDX1 → DoxA pathway for the hydroxylation of the daunorubicin, and indicate that the usage of a native redox partner may increase daunorubicin-derived doxorubicin production due to the inclusion of DoxA.

  • homology modeling and in vitro analysis for characterization of Streptomyces Peucetius cyp157c4
    Journal of Microbiology and Biotechnology, 2015
    Co-Authors: Hemraj Rimal, Jong Hwa Jang, Sangcheol Yu, Taejin Oh
    Abstract:

    : In this study, we tried to characterize Streptomyces Peucetius CYP157C4 with homology modeling using three cytochrome P450 (CYP) structures (CYP157C1, CYP164A2, and CYP107L1), having discovered that CYP157C4 lacks the ExxR motif that was considered invariant in all CYPs. We used Discovery Studio 3.5 to build our model after first assessing the stereochemical quality and side-chain environment, and a 7-ethoxycoumarin substrate was docked into the final model. The model-substrate complex allowed us to identify functionally important residues and validate the active-site architecture. We found a distance of 4.56 A between the 7-ethoxycoumarin and the active site of the heme, and cloning and an in vitro assay of the CYP157C4 showed the dealkylation of the substrate. Since the details regarding this group of CYP structures are still unknown, the findings of this study may provide elucidation to assist with future efforts to find a legitimate substrate.

  • disruption of negative regulators sp_nsda and sp_nsdb in Streptomyces Peucetius causes doxorubicin overproduction
    African Journal of Microbiology Research, 2014
    Co-Authors: Kyounghee Kwon, Jong Hwa Jang, Taejin Oh
    Abstract:

    Pathway-specific regulatory genes generally participate in the secondary metabolites-related biosynthesis process. The genes nsdA and nsdB were reported to have a negative effect on the production of actinorhodin, prodigiosin and calcium-dependent antibiotic from Streptomyces coelicolor. In this study, we searched for similar genes in the Streptomyces Peucetius genome, the doxorubicin producer. Amino acid sequence similarity between SC_NsdA and SP4635 (SP_NsdA) was 88.1%, and between SC_NsdB and SP1750 (SP_NsdB) was 78.4%. High performance liquid chromatography (HPLC) analysis revealed that the disruption of SP_nsdA and SP_nsdB significantly increased doxorubicin production by 2.07 and 1.74-fold, respectively. The SP_nsdA and SP_nsdB disruption mutants produced more yellow pigment and early aerial mycelium than did the original wild-type strain. These results show that SP_nsdA and SP_nsdB negatively affected doxorubicin production and morphological differentiation in S. Peucetius. Key words: Doxorubicin, gene disruption, negative regulator, secondary metabolite, Streptomyces Peucetius.

  • characterization of dephosphocoenzyme a kinase from Streptomyces Peucetius atcc27952 and its application for doxorubicin overproduction
    Journal of Microbiology and Biotechnology, 2014
    Co-Authors: Hemraj Rimal, Taejin Oh
    Abstract:

    : Dephosphocoenzyme A (CoaE) catalyzes the last step in the biosynthesis of the cofactor coenzyme A. In this study, we report the identification and application of CoaE from Stretomyces Peucetius ATCC27952. After expression of coaE, the protein was found to have a molecular mass of 28.6 kDa. Purification of the His-tagged fused CoaE protein was done by immobilized metal-affinity chromatography, and then in vitro enzymatic coupling assay was performed. The increasing NADH consumption with time shed light on the phosphorylating activity of CoaE. Furthermore, the overexpression of coaA and coaE independently under the ermE(*) promoter in the doxorubicin -producing wild type strain, resulted in 1.4- and 1.5-fold enhancements in doxorubicin production, respectively. In addition, the overexpression of both genes together showed a 2.1-fold increase in doxorubicin production. These results established a positive role for secondary metabolite production from Streptomyces Peucetius.

Ranjan Prasad - One of the best experts on this subject based on the ideXlab platform.

  • dnri of Streptomyces Peucetius binds to the resistance genes drrab and drrc but is activated by daunorubicin
    Journal of Basic Microbiology, 2017
    Co-Authors: Francis Prija, Padmanabhan Srinivasan, Karuppasamy Kattusamy, Ranjan Prasad
    Abstract:

    : The master regulator, DnrI of Streptomyces Peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

  • drrc protein of Streptomyces Peucetius removes daunorubicin from intercalated dnri promoter
    Microbiological Research, 2017
    Co-Authors: Francis Prija, Ranjan Prasad
    Abstract:

    Abstract DrrC is a DNA-binding protein of Streptomyces Peucetius that provides self-resistance against daunorubicin, the antibiotic produced by the organism. DrrC was expressed in E.coli and purified by using N-terminal MBP-tag which retained DNA-binding property in spite of the tag. Mobility shift assay confirmed the interaction of 313 bp DNA that has the dnrI promoter, daunorubicin and MBP-DrrC in the presence of ATP. Biotinylated and immobilized 313 bp DNA was intercalated with daunorubicin to observe the release of the drug when MBP-DrrC is allowed to act on the DNA. The release of daunorubicin was recorded by absorption and fluorescence spectroscopy. The experiments proved that daunorubicin was released from DNA in the presence of MBP-DrrC. Fluorescence emission of daunorubicin had a maximum peak at 591 nm. However, emission spectrum of released daunorubicin showed hypochromism with a maximum peak at 584 nm that is possibly because it is in complex with MBP-DrrC. We propose that DrrC naturally binds at intercalated sites to eject daunorubicin; in the process both drug and protein are dislodged from DNA. Like UvrA, DrrC possibly scans the DNA for intercalated daunorubicin. When it encounters daunorubicin, DrrC dislodges it, thereby allowing DNA replication and transcription to go on unhindered. Thus a novel self resistance mechanism by DNA repair is mediated by DrrC.

  • partial loss of self resistance to daunorubicin in drrd mutant of Streptomyces Peucetius
    Biochemical Engineering Journal, 2015
    Co-Authors: Kattusamy Karuppasamy, Padmanabhan Srinivasan, Balasubramaniem Ashokkumar, Rahul Tiwari, Karuppiah Kanagarajadurai, Ranjan Prasad
    Abstract:

    Abstract Self-resistance is a key element for survival of antibiotic producing Streptomyces . Self-resistance in Streptomyces Peucetius is conferred by drrA , drrB and drrC genes to survive the toxicity of daunorubicin produced by the organism. The fourth gene is drrD , which is found in drrAB operon and was reported as a possible self-resistance gene. In this study, a drrD mutant is constructed, which showed loss of self-resistance partially to daunorubicin. This study establishes drrD gene as self-resistance gene and along with drrA , drrB and drrC it forms the full complement of self-resistance that protects S. Peucetius from the toxicity of daunorubicin. Bioinformatics analysis of DrrD protein showed that it has N-terminal FAD binding domain. It belongs to a family of proteins that does oxidoreductase and transferase activity. DrrD shares high similarity with FAD binding AknOx protein of S. galilaeus , the aclacinomycin producer. DrrD protein was expressed and the partially purified protein was able to bind to FAD. AknOx binds to sugar moiety of aclacinomycin and modifies it to a temporary inactive form and similar role for DrrD is discussed.

  • biophysical characterization of in vitro bound Streptomyces Peucetius daunorubicin serine protease complex
    International Journal of Biological Macromolecules, 2014
    Co-Authors: Rashmi Dubey, Ranjan Prasad
    Abstract:

    Abstract A serine protease of Streptomyces Peucetius is found in association with daunorubicin in the culture filtrate and co-purifies as a complex as reported earlier by us (Dubey et al., 2013). The same protease was purified without drug attachment from dpsA− mutant of S. Peucetius, which does not produce daunorubicin. Drug–protein complex was made in vitro by mixing daunorubicin and the protease. Spectral analysis and circular dichroism (CD) analysis were employed to determine the interaction between daunorubicin and the protease. Our study showed that interaction of daunorubicin with the protease affects the spectral characteristics of the drug and changes the secondary structure of the protein. Thin layer chromatography (TLC) analysis showed that the drug–protein interaction results in partial conversion of the drug to aglyconic form. The complex formation implies sequestration of the drug when it attains potentially lethal level in the extracellular milieu of S. Peucetius culture.

  • daunorubicin forms a specific complex with a secreted serine protease of Streptomyces Peucetius
    World Journal of Microbiology & Biotechnology, 2014
    Co-Authors: Rashmi Dubey, Kuppamuthu Dharmalingam, Karuppasamy Kattusamy, Ranjan Prasad
    Abstract:

    Daunorubicin forms specific complex with an extracellular protease in the Streptomyces Peucetius culture. The drug-protein complex co-migrates in non-denaturing PAGE as a red band. De novo peptide sequencing by nano-LC–ESI–MS/MS and MASCOT analysis identified the daunorubicin binding protein as serine protease precursor. The same protease precursor was purified sans the daunorubicin, from the mutant named ΔDPSAmut, which is deficient in daunorubicin production. Daunorubicin was added to ΔDPSAmut culture and the protease readily formed the daunorubicin-protease complex. Ability of serine protease precursor to form a selective complex with daunorubicin was confirmed by this study. Selective binding of protease to daunorubicin was seen as self-resistance determinant for the organism to survive toxic levels of the drug outside the cell. Daunorubicin-protease complex placed on S. Peucetius lawn did not produce clearing zone around it, whereas daunorubicin purified from the complex did produce the clearing zone. Thereby it is concluded that the protease sequesters daunorubicin to prevent its entry into cells. Sequestration of daunorubicin by extracellular protease helps the organism to maintain a steady state sub-inhibitory level of drug around the cells. A new self-resistance determinant is reported here.

Sergio Sanchez - One of the best experts on this subject based on the ideXlab platform.

  • functional analysis of the glcp promoter in Streptomyces Peucetius var caesius
    Applied Biochemistry and Biotechnology, 2015
    Co-Authors: Alba Romero, Jae Kyung Sohng, Niranjan Koirala, Romina Rodriguezsanoja, Beatriz Ruiz, Sergio Sanchez
    Abstract:

    In Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. Glucose is mainly transported by GlcP, a membrane protein encoded by glcp. In Streptomyces coelicolor, this protein is encoded by sco5578. However, there is little information about the physiology of the GlcP promoter in Streptomyces. The aim of the present work was to clone and perform a functional analysis of the sp7066 promoter (ortholog of sco5578) from Streptomyces Peucetius var. caesius. Hydrophobicity and cellular location analysis of the putative amino acid sequence of the cloned gene predicted SP7066 would be a membrane protein with a topology of six plus six transmembrane segments interrupted by a large cytoplasmic loop. In silico analysis of the upstream region of the sp7066 transcription initiation site predicted the sequences 5′-AGGAATAGT-3′ and 5′-TTGACT-3′ for regions -10 and -35 of sp7066 promoter. To reflect sp7066 expression, the promoter sequence was amplified, subcloned, and fused to the egfp reporter gene. Immunoblot analysis revealed that D-glucose and its analog 2-deoxyglucose were able to induce sp7066 expression. This effect was not modified by the presence of equimolar concentrations of D-galactose or N-acetylglucosamine. No expression of egfp was detected with the use of other carbon sources such as L-arabinose, D-fructose, and glycerol. Based on these analyses, we conclude that D-glucose is a preferred carbon source in S. Peucetius var. caesius and that the sp7066 expression product, a putative non-PTS glucose permease, likely is a H+/symporter, localized to the membrane, and shows a strong specificity for D-glucose for inducing expression.

  • glucose kinases from Streptomyces Peucetius var caesius
    Applied Microbiology and Biotechnology, 2014
    Co-Authors: Beatriz Ruizvillafan, Romina Rodriguezsanoja, Guillermo Aguilarosorio, Guillermo Gosset, Sergio Sanchez
    Abstract:

    Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces Peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. Peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. Peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. Peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX(4)SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins.

  • sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces Peucetius var caesius
    Applied Microbiology and Biotechnology, 2005
    Co-Authors: Silvia Guzman, Romina Rodriguezsanoja, Laura Escalante, Itzel Ramos, Elizabeth Langley, Beatriz Ruiz, Elizabeth Moreno, Sergio Sanchez
    Abstract:

    Streptomyces Peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. Peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. Peucetius var. caesius.

  • biochemical characterization of the glucose kinase from Streptomyces coelicolor compared to Streptomyces Peucetius var caesius
    Research in Microbiology, 2005
    Co-Authors: Iveta Imriskova, Romina Rodriguezsanoja, Elizabeth Langley, Roberto Arreguinespinosa, Silvia Guzman, Sergio Sanchez
    Abstract:

    Abstract Glucose kinase (Glk) from Streptomyces coelicolor was purified, characterized biochemically and kinetically, and compared to Glk from Streptomyces Peucetius var. caesius. In both Streptomyces, 96% of the enzyme activity was detected in the cytosolic fraction. The use of a Glk activity gel showed no isoforms of the enzyme in extracts from these microorganisms. The purified S. coelicolor Glk was stable in its tetrameric form, unlike the purified enzyme from S. Peucetius var. caesius which easily dissociated into dimers. Tetramer dissociation was prevented by 100 mM d -glucose; however, this effect was not observed with other sugars. The optimum pH and the pI are practically identical for both enzymes. Maximum activity was found at a lower temperature for S. coelicolor Glk (33 compared to 42 °C) and its activity was apparently less stable at higher temperatures. Its activation energy was also 40% lower than that of S. Peucetius var. caesius. The kinetic mechanism appears to follow a rapid equilibrium-ordered Bi–Bi sequential mechanism in both microbial enzymes, where Glk first binds glucose and then the MgATP2− complex, to form a ternary complex (enzyme d -glucose-MgATP−2). The K m values for d -glucose and MgATP2− were 1.4, 0.5 mM and 1.6, 0.8 mM for the S. coelicolor and S. Peucetius var. caesius Glks, respectively. However V max of S. coelicolor Glk was higher. In conclusion, the S. coelicolor Glk showed a more stable tetrameric form with better affinity for its substrates and higher V max , suggesting greater catalytic efficiency.

  • glucose kinase alone cannot be responsible for carbon source regulation in Streptomyces Peucetius var caesius
    Research in Microbiology, 2004
    Co-Authors: Itzel Ramos, Romina Rodriguezsanoja, Sergio Sanchez, Laura Escalante, Iveta Imriskova, Silvia Guzman, Elizabeth Langley
    Abstract:

    Abstract Using an antibiotic enrichment procedure, eight mutants of Streptomyces Peucetius var. caesius were isolated for their sensitivity to the glucose analogue 2-deoxyglucose (DOG), from a DOG-resistant strain (Dog R ). These mutants (Dog S ) and their parent strain were examined for growth sensitivity to DOG, glucose kinase (Glk) activity, glucose uptake, and sensitivity to repression by glucose and other catabolites derived from it. No correlation was found between Glk levels or glucose uptake and carbon catabolite repression (CCR) in these strains. However, the ratio of glucose uptake to Glk activity, and thus the flux through glycolysis, seemed responsible for this effect. Among several products of glucose catabolism tested, fructose-1,6-bis-phosphate and phosphoenolpyruvate showed significant repression of anthracycline formation. These compounds also reduced anthracycline formation in a Dog R mutant insensitive to glucose repression. Our data suggest that Glk alone is not sufficient to elicit CCR in this microorganism, and gives the first physiological evidence supporting the hypothesis that some products of glucose catabolism are involved in CCR in Streptomyces .

C R Hutchinson - One of the best experts on this subject based on the ideXlab platform.