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Indian Journal of Microbiology Dec 2021Anthracycline drugs have multifunctional molecular structures, and small changes in the structure of the glycosyls around the chromophore affect their mechanism of... (Review)
Review
Anthracycline drugs have multifunctional molecular structures, and small changes in the structure of the glycosyls around the chromophore affect their mechanism of action, pharmacokinetics, toxicity, anti-tumor activity, and many other significant parameters. has a similar function to but inverse stereoselectivity. encoding amino transferase was substituted for for the purpose of obtaining nogalamycin analogues. We inactivated the gene encoding an aminotransferase responsible for the formation of nogalamine and introduced the gene encoding an aminotransferase responsible for the formation of daunosamine. We obtained the recombinant strain mLMX-3-100, in which the production of nogalamycin was disrupted. Interestingly, contrary to our predictions, no epi-nogalamycin was produced; nevertheless, the present study shows that the gene is necessary for the proper functioning of the nogalamycin biosynthesis pathway. These data may provide a reference for further illustration of nogalamycin biosynthesis and its modification by way of combinatorial biosynthesis.biosynthesis and its modification by way of combinatorial biosynthesis.
PubMed: 34744202
DOI: 10.1007/s12088-021-00941-7 -
Malaria Journal Jul 2022Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial...
BACKGROUND
Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites.
METHODS
In this study, a P. falciparum 5' to 3' DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated.
RESULTS
The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3' or 5' end and a linear substrate with 3' end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC = 0.1 ± 0.002 µM).
CONCLUSION
Partial purification and characterization of 5'-3' DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3' to 5' DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.
Topics: Anthracyclines; Antimalarials; DNA; DNA Helicases; Humans; Malaria, Falciparum; Nogalamycin; Plasmodium falciparum
PubMed: 35821133
DOI: 10.1186/s12936-022-04238-y -
Acta Crystallographica. Section F,... Mar 200912-deoxy-nogalonic acid oxygenase (SnoaB) catalyzes the oxygenation of 12-deoxy-nogalonic acid at position 12 to yield nogalonic acid, which is one of the steps in the...
12-deoxy-nogalonic acid oxygenase (SnoaB) catalyzes the oxygenation of 12-deoxy-nogalonic acid at position 12 to yield nogalonic acid, which is one of the steps in the biosynthesis of the polyketide nogalamycin in Streptomyces nogalater. SnoaB belongs to a family of small cofactor-free oxygenases which carry out oxygenation reactions without the aid of any prosthetic group, cofactor or metal ion. Recombinant SnoaB was crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 58.8, b = 114.1, c = 49.5 A, and these crystals diffracted to 2.4 A resolution. Recombinant SnoaB does not contain any methionine residues and three double mutants were designed and produced for the preparation of selenomethionine-substituted samples. The selenomethionine-substituted mutant F40M/L89M crystallized in the same space group as the native enzyme.
Topics: Amino Acid Sequence; Catalysis; Coenzymes; Crystallization; Crystallography, X-Ray; Mixed Function Oxygenases; Molecular Sequence Data; Mutant Proteins; Nogalamycin; Selenomethionine; Sequence Alignment; Streptomyces
PubMed: 19255477
DOI: 10.1107/S1744309109001389 -
ACS Chemical Biology Sep 2018Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units....
Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C-C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.
Topics: Amino Sugars; Anthracyclines; Antibiotics, Antineoplastic; Biocatalysis; Enzymes, Immobilized; Glycosylation; Nogalamycin; Streptomyces; Thymine Nucleotides
PubMed: 30114358
DOI: 10.1021/acschembio.8b00658 -
Plant Signaling & Behavior Mar 2011Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3' to 5' DNA...
Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3' to 5' DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities [Plant Mol. Biol. 2010; DOI: 10.1007/s11103-010-9675-7]. Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 μM, respectively) and ATPase (apparent Ki values of 100 and 17 μM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding.
Topics: Adenosine Triphosphatases; Camptothecin; Cisplatin; DNA Helicases; Dactinomycin; Daunorubicin; Ellipticines; Enzyme Inhibitors; Etoposide; Intercalating Agents; Mitoxantrone; Nogalamycin; Novobiocin; Pisum sativum; Plant Proteins
PubMed: 21336027
DOI: 10.4161/psb.6.3.13355 -
Biochemistry and Molecular Biology... Sep 2016The "Streptomyces genetics and secondary metabolism" laboratory course gives an introduction to the versatile soil dwelling Gram-positive bacteria Streptomyces and their...
The "Streptomyces genetics and secondary metabolism" laboratory course gives an introduction to the versatile soil dwelling Gram-positive bacteria Streptomyces and their secondary metabolism. The course combines genetic modification of Streptomyces; growing of the strain and protoplast preparation, plasmid isolation by alkaline lysis and phenol precipitation, digestions, and ligations prior to protoplast transformation, as well as investigating the secondary metabolites produced by the strains. Thus, the course is a combination of microbiology, molecular biology, and chemistry. After the course the students should understand the relationship between genes, proteins, and the produced metabolites. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(5):492-499, 2016.
Topics: Bacterial Proteins; Biomedical Research; Chemistry; Chromatography, Thin Layer; Curriculum; Educational Measurement; Genetics; Humans; Laboratories; Microbiology; Models, Biological; Plasmids; Problem-Based Learning; Secondary Metabolism; Streptomyces; Students; Transformation, Bacterial
PubMed: 27192442
DOI: 10.1002/bmb.20970 -
The Journal of Biological Chemistry May 2004Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in...
Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5'-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 microM. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5'-CCCTT/G instead of 5'-CCCTT/A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.
Topics: Base Pairing; Base Sequence; DNA Adducts; DNA Topoisomerases, Type I; Hydrolysis; Intercalating Agents; Models, Molecular; Molecular Sequence Data; Nogalamycin; Nucleic Acid Conformation; Phenanthrenes; Vaccinia virus
PubMed: 15044474
DOI: 10.1074/jbc.M401203200 -
Advanced Science (Weinheim,... Apr 2024Tumor-associated macrophages (TAMs) play a crucial role in promoting tumor growth and dissemination, motivating a search for key targets to interfere with the activation...
Tumor-associated macrophages (TAMs) play a crucial role in promoting tumor growth and dissemination, motivating a search for key targets to interfere with the activation of TAMs or reprogram TAMs into the tumor-suppressive type. To gain insight into the mechanisms of macrophage polarization, a designed co-culture system is established, allowing for the education of macrophages in a manner that closely mimics the intricacies of TAMs in the tumor immune microenvironment (TIME). Through database mining, exosomal miR-1246 is identified and is then validated. Exosomal miR-1246-driven polarization of TAMs disrupts the infiltration and function of CD8 T cells. Mechanically, the amassment of exosomal miR-1246 stems from TUT7-mediated degradation of small noncoding RNA, a process stabilized by SNRPB, but not the precursor of miR-1246. Moreover, an Exo-motif is present in the exosomal miR-1246 sequence, enabling it to bind with the exosomal sorting protein hnRNPA2B1. RNA-seq analysis reveals that exogenous miR-1246 modulates the polarization of TAMs at a post-transcriptional level, emphasizing the pivotal role of the NLRP3 in macrophage polarization. In conclusion, the findings underscore the importance of exosomal miR-1246 as a trigger of macrophage reprogramming and uncover a novel mechanism for its enhanced presence in the TIME.
Topics: Tumor-Associated Macrophages; Menogaril; CD8-Positive T-Lymphocytes; MicroRNAs; Macrophages
PubMed: 38342611
DOI: 10.1002/advs.202304222 -
RNA (New York, N.Y.) Nov 2022Metazoan histone mRNAs are the only cellular eukaryotic mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. SLBP is bound to the 3' end of...
Metazoan histone mRNAs are the only cellular eukaryotic mRNAs that are not polyadenylated, ending instead in a conserved stem-loop. SLBP is bound to the 3' end of histone mRNAs and is required for translation of histone mRNA. The expression of histone mRNAs is tightly cell-cycle regulated. A major regulatory step is rapid degradation of histone mRNA at the end of S-phase or when DNA synthesis is inhibited in S-phase. 3'hExo, a 3' to 5' exonuclease, binds to the SLBP/SL complex and trims histone mRNA to 3 nt after the stem-loop. Together with a terminal uridyl transferase, 3'hExo maintains the length of the histone mRNA during S-phase. 3'hExo is essential for initiating histone mRNA degradation on polyribosomes, initiating degradation into the 3' side of the stem-loop. There is extensive uridylation of degradation intermediates in the 3' side of the stem when histone mRNA is degraded. Here, we knocked out TUT7 and 3'hExo and we show that both modification of histone mRNA during S-phase and degradation of histone mRNA involve the interaction of 3'hExo, and a specific TUTase, TENT3B (TUT7, ZCCHC6). Knockout of 3'hExo prevents the initiation of 3' to 5' degradation, stabilizing histone mRNA, whereas knockout of TUT7 prevents uridylation of the mRNA degradation intermediates, slowing the rate of degradation. In synchronized 3'hExo KO cells, histone mRNA degradation is delayed, but the histone mRNA is degraded prior to mitosis by a different pathway.
Topics: Animals; Humans; Histones; Menogaril; HeLa Cells; RNA Stability; RNA, Messenger; mRNA Cleavage and Polyadenylation Factors
PubMed: 36041871
DOI: 10.1261/rna.079233.122 -
The FEBS Journal Jul 2020Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of...
Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2-oxoglutarate-dependent mono-oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2-C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C-terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2-C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents. ENZYMES: EC number 1.14.11.
Topics: Bacterial Proteins; Biosynthetic Pathways; Evolution, Molecular; Genetic Engineering; Nogalamycin; Nonheme Iron Proteins; Protein Conformation; Streptomyces
PubMed: 31876382
DOI: 10.1111/febs.15192