-
Chembiochem : a European Journal of... Nov 2011Mix'n'match: Enzymatic total synthesis of TDP-D-olivose was achieved, starting from TDP-4-keto-6-deoxy-D-glucose, by combining three pathway enzymes with one...
Mix'n'match: Enzymatic total synthesis of TDP-D-olivose was achieved, starting from TDP-4-keto-6-deoxy-D-glucose, by combining three pathway enzymes with one cofactor-regenerating enzyme. The results also revealed that MtmC is a bifunctional enzyme that can perform a 4-ketoreduction necessary for D-olivose biosynthesis besides the previously found C-methyltransfer for D-mycarose biosynthesis.
Topics: Deoxy Sugars; Enzymes; Glucose; Nucleoside Diphosphate Sugars; Oxidation-Reduction; Plicamycin; Thymine Nucleotides
PubMed: 21960454
DOI: 10.1002/cbic.201100540 -
Nucleic Acids Research 2005Mith (mithramycin) forms a 2:1 stoichiometry drug-metal complex through the chelation with Fe(II) ion as studied using circular dichroism spectroscopy. The binding...
Mith (mithramycin) forms a 2:1 stoichiometry drug-metal complex through the chelation with Fe(II) ion as studied using circular dichroism spectroscopy. The binding affinity between Mith and Fe(II) is much greater than other divalent metal ions, including Mg(II), Zn(II), Co(II), Ni(II) and Mn(II). The [(Mith)2-Fe(II)] complex binds to DNA and induces a conformational change of DNA. Kinetic analysis of surface plasmon resonance studies revealed that the [(Mith)2-Fe(II)] complex binds to DNA duplex with higher affinity compared with the [(Mith)2-Mg(II)] complex. A molecular model of the Mith-DNA-Metal(II) complex is presented. DNA-break assay showed that the [(Mith)2-Fe(II)] complex was capable of promoting the one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide. Intracellular Fe(II) assays and fluorescence microscopy studies using K562 indicated that this dimer complex maintains its structural integrity and permeates into the inside of K562 cells, respectively. The [(Mith)2-Fe(II)] complex exhibited higher cytotoxicity than the drug alone in some cancer cell lines, probably related to its higher DNA-binding and cleavage activity. Evidences obtained in this study suggest that the biological effects caused by the [(Mith)2-Fe(II)] complex may be further explored in the future.
Topics: Antibiotics, Antineoplastic; Base Sequence; Cations, Divalent; Cell Line, Tumor; Cell Membrane Permeability; Circular Dichroism; DNA; Ferrous Compounds; Humans; Iron Chelating Agents; K562 Cells; Metals; Models, Molecular; Nucleic Acid Conformation; Plasmids; Plicamycin; Surface Plasmon Resonance
PubMed: 15741187
DOI: 10.1093/nar/gki276 -
The Journal of Clinical Investigation Nov 1991The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility...
The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.
Topics: Base Sequence; Drug Resistance; Gene Expression Regulation, Enzymologic; Humans; Methotrexate; Molecular Sequence Data; Plicamycin; Promoter Regions, Genetic; Sp1 Transcription Factor; Tetrahydrofolate Dehydrogenase; Transcription, Genetic
PubMed: 1834700
DOI: 10.1172/JCI115474 -
Biochemistry Jul 1993We have characterized the NMR parameters for the complexes formed by the Mg(2+)-coordinated mithramycin dimer with self-complementary d(T-G-G-C-C-A) and d(T-C-G-C-G-A)... (Comparative Study)
Comparative Study
We have characterized the NMR parameters for the complexes formed by the Mg(2+)-coordinated mithramycin dimer with self-complementary d(T-G-G-C-C-A) and d(T-C-G-C-G-A) duplexes. The solution structure of the latter complex has been determined using a combined NMR-molecular dynamics study including relaxation matrix refinement. The Mg(2+)-coordinated mithramycin dimer-d(T-C-G-C-G-A) complex exhibits a 2-fold center of symmetry with the divalent cation coordinated aglycons positioned opposite the central (G3-C4).(G3-C4) segment such that the aglycon C8 hydroxyl oxygens form symmetrical sequence-specific hydrogen bonds to guanine amino protons in the complex. The C-D-E trisaccharide segments of each monomer in the mithramycin dimer adopt extended conformations, are positioned inside the minor groove, and are directed toward either end of the duplex. The C-D saccharide component of one monomer and the aglycon of the other monomer in the mithramycin dimer share a widened minor groove with the hydrophobic edges of the C and D sugars interacting with individual strands of the duplex. The E-sugar ring is positioned in the floor of the minor groove, and its hydroxyl-bearing face interacts with both strands of the duplex through hydrogen-bonding and hydrophobic intermolecular interactions. The A-B disaccharide and the hydrophilic side chain form intermolecular contacts with the sugar-phosphate backbone in the complex. The antiparallel alignment of divalent cation coordinated monomers in the mithramycin dimer results in the two outwardly directed C-D-E trisaccharide segments generating a right-handed continuous hexasaccharide domain that spans six base pairs in the minor groove of the duplex. The solution structure of the mithramycin dimer-DNA complex reported in this study and the solution structure of the chromomycin dimer-DNA complex reported previously [Gao, X., Mirau, P., & Patel, D. J. (1992) J. Mol. Biol. 223, 259-279] show global similarities, as well as local differences that are of interest. All four nucleotides in the tetranucleotide segment of the duplex centered about the sequence-specific (G-C).(G-C) step adopt A-DNA sugar puckers and glycosidic torsion angles in the chromomycin dimer-DNA complex, while only the central cytidine adopts an A-DNA sugar pucker and glycosidic torsion angle in the mithramycin dimer-DNA complex.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Base Sequence; DNA; Magnesium; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Plicamycin; Structure-Activity Relationship
PubMed: 8329387
DOI: 10.1021/bi00077a012 -
Chemico-biological Interactions Aug 2014DIG-MSK (demycarosyl-3D-β-D-digitoxosyl-mithramycin SK) is a recently isolated compound of the mithramycin family of antitumor antibiotics, which includes mithramycin A...
DIG-MSK (demycarosyl-3D-β-D-digitoxosyl-mithramycin SK) is a recently isolated compound of the mithramycin family of antitumor antibiotics, which includes mithramycin A (MTA) and mithramycin SK (MSK). Here, we present evidence that the binding of DIG-MSK to DNA shares the general features of other mithramycins such as the preference for C/G-rich tracts, but there are some differences in the strength of binding and the DNA sequence preferentially recognized by DIG-MSK. We aimed at gaining further insights into the DIG-MSK mechanism of action by direct comparison with the effects of the parental MTA. Similar to MTA, MSK and DIG-MSK accumulated rapidly in A2780, IGROV1 and OVCAR3 human ovarian cancer cell lines, and DIG-MSK was a potent inhibitor of both basal and induced expression of an Sp1-driven luciferase vector. This inhibitory activity was confirmed for the endogenous Sp1 gene and a set of Sp-responsive genes, and compared to that of MTA and MSK. Furthermore, DIG-MSK was stronger than MTA as inhibitor of Sp3-driven transcription and endogenous Sp3 gene expression. Differences in the effects of MTA, MSK and DIG-MSK on gene expression may have a large influence on their biological activities.
Topics: Antibiotics, Antineoplastic; Binding Sites; Cell Line, Tumor; Female; GPI-Linked Proteins; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Ovarian Neoplasms; Plicamycin; RNA; Real-Time Polymerase Chain Reaction; Sp1 Transcription Factor; Spectrometry, Fluorescence; Transcription, Genetic
PubMed: 24907531
DOI: 10.1016/j.cbi.2014.05.019 -
Journal of Molecular Biology Sep 1995Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore....
Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg(2+)-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C).(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C).(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA 10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A).(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.
Topics: Base Composition; Base Sequence; Binding Sites; Chromomycins; Computer Graphics; DNA; Intercalating Agents; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Molecular Structure; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Plicamycin; Protons; Trisaccharides
PubMed: 7666419
DOI: 10.1006/jmbi.1995.0464 -
Journal of Controlled Release :... Dec 2001Arterial restenosis is responsible for the high failure rates of vascular reconstruction procedures. Local sustained drug delivery has shown promise in the prevention of...
Arterial restenosis is responsible for the high failure rates of vascular reconstruction procedures. Local sustained drug delivery has shown promise in the prevention of restenosis. The drug release rate from mithramycin-loaded EVA matrices (0.1%) was evaluated, and their antirestenotic effect was studied in the rat carotid model and rabbit model of vascular grafts. The modulation of c-myc expression by mithramycin treatment was examined by immunohistochemistry in the rat carotid model. The proliferative response of injured rat arteries was studied by bromdeoxyuridine (BrdU) immunostaining. The impact of mithramycin treatment on vasomotor responses of the venous segments grafted into arterial circulation was studied ex vivo using vasoreactive compounds. Mithramycin was released exponentially from EVA matrices in PBS. Matrices co-formulated with PEG-4600 revealed enhanced release kinetics. The perivascular implantation of drug-loaded EVA-PEG matrices led to 50% reduction of neointimal formation, and reduced the c-myc expression and BrdU labeling in comparison to control implants. Decreased sensitivity of mithramycin-treated grafts to serotonin-induced vasoconstriction was observed. Local perivascular mithramycin treatment limits the functional alteration caused by the grafting of venous segments in high-pressure arterial environment, and potently inhibits stenosis secondary to grafting and angioplasty injury. The antirestenotic effect is associated with reduced c-myc expression and with subsequent decrease in SMC proliferation.
Topics: Animals; Carotid Artery, Common; Catheterization; Cells, Cultured; Drug Delivery Systems; Graft Occlusion, Vascular; Growth Inhibitors; Jugular Veins; Male; Muscle, Smooth, Vascular; Plicamycin; Rabbits; Rats; Swine; Tunica Intima
PubMed: 11733085
DOI: 10.1016/s0168-3659(01)00472-2 -
Journal of Inorganic Biochemistry Jan 2015Pathogenic bacteria that are resistant to β-lactam antibiotics mostly utilize serine β-lactamases to degrade the antibiotics. Current studies have shown that different...
Pathogenic bacteria that are resistant to β-lactam antibiotics mostly utilize serine β-lactamases to degrade the antibiotics. Current studies have shown that different subclasses of metallo β-lactamases (E[MBL]) are involved in the defense mechanism of drug resistant bacteria. Here we report that the Zn(2+) containing subclass B1 E[MBL] from Bacillus cereus binds to a naturally occurring anti-cancer drug mithramycin (MTR). Spectroscopic (CD and fluorescence) and isothermal titration calorimetry studies show that MTR forms a high affinity complex with the Zn(2+) ion containing E[MBL]. Abolished interaction of MTR with apo E[MBL] suggests that the formation of this high affinity complex occurs due to the potential of MTR to bind bivalent metal ions like Zn(2+). Furthermore, CD spectroscopy, dynamic light scattering and differential scanning calorimetry studies indicate that the strong association with sub-micromolar dissociation constant leads to an alteration in the enzyme conformation at both secondary and tertiary structural levels. The enzyme activity decreases as a consequence to this conformational disruption arising from the formation of a ternary complex involving MTR, catalytic Zn(2+) and the enzyme. Our results suggest that the naturally occurring antibiotic MTR, a generic drug, has the potential as an E[MBL] inhibitor.
Topics: Antibiotics, Antineoplastic; Bacillus cereus; Bacterial Proteins; Plicamycin; Zinc; beta-Lactamases
PubMed: 25450021
DOI: 10.1016/j.jinorgbio.2014.10.001 -
Journal of Bacteriology Jan 1999Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The...
Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Carbohydrate Sequence; Chromosome Mapping; Chromosomes, Bacterial; Consensus Sequence; DNA, Bacterial; Genes, Regulator; Molecular Sequence Data; Molecular Structure; Multigene Family; Open Reading Frames; Plicamycin; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Sequence Alignment; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Streptomyces; Trans-Activators
PubMed: 9882681
DOI: 10.1128/JB.181.2.642-647.1999 -
Calcified Tissue Research Dec 1978Mithramycin suppresses bone resorption. Its effect on the synthesis and release of beta-glucuronidase (a referent for lysosomal enzymes) in mouse calvarial explants was...
Mithramycin suppresses bone resorption. Its effect on the synthesis and release of beta-glucuronidase (a referent for lysosomal enzymes) in mouse calvarial explants was studied in an in vitro culture system. A newly described medium (designated as KT medium) was introduced in this specific study. Mithramycin initially inhibited the release of beta-glucuronidase into the medium and resulted in an ultimate accumulation of this enzyme in the bone. These results suggest that inhibition of bone resorption by mithramycin may be attributed to interference in release of lysosomal enzymes from bone cells.
Topics: Animals; Bone Resorption; Bone and Bones; Cells, Cultured; Enzyme Induction; Glucuronidase; Mice; Parathyroid Hormone; Plicamycin; Vitamin A
PubMed: 155493
DOI: 10.1007/BF02013260