Discovery of novel septacidin congeners from a high yield heterologous expression strain Streptomyces albus 1597.

Septacidin is an adenine nucleoside antibiotic with antifungal and antitumor activities. During the efforts to construct a better septacidin producer, we obtained a high yield strain S. albus 1597 by putting the biosynthetic gene cluster (BGC) of septacidin under the control of the constitutive strong promoter ermE*. S. albus 1597 could produce new septacidin congeners SEP-538 and SEP-552 with shorter fatty acyl chains. Moreover, SEP-624 with an unprecedented hydroxylated fatty acyl chain was also isolated from this titre improved strain, enriching the diversity of septacidins. SEP-552 showed moderate inhibitory effects against Epidermophyton floccosum 57312 with MIC value 62.5 µM, while SEP-538 and SEP-624 only exhibited weak antifungal activities. The structure-activity relationship investigation revealed that the antifungal activity of septacidins is significantly influenced by the length of and the decoration on their fatty acyl chains.

Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate.

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.

Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides.

The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.

Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking.

BACKGROUND: Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC-MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. RESULTS: The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. CONCLUSIONS: A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the "cryptic" herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

Identification and Interrogation of the Herbicidin Biosynthetic Gene Cluster: First Insight into the Biosynthesis of a Rare Undecose Nucleoside Antibiotic.

Herbicidins are adenosine-based nucleoside antibiotics with an unusual tricyclic undecose core decorated with a (5-hydroxy)tiglyl moiety. Feeding studies are herein reported demonstrating that the tricyclic core is derived from d-glucose and d-ribose, whereas the tiglyl moiety is derived from an intermediate of l-isoleucine catabolism. Identification of the gene cluster for herbicidin A biosynthesis in Streptomyces sp. L-9-10 as well as its verification by heterologous expression in a nonproducing host are described, and the results of in vitro characterization of a carboxyl methyltransferase encoded in the cluster, Her8, are presented. Based on these observations, a biosynthetic pathway is proposed for herbicidins.

Metabolomic profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cystic fibrosis cells.

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.

Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine.

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.

The case for 8,5'-cyclopurine-2'-deoxynucleosides as endogenous DNA lesions that cause neurodegeneration in xeroderma pigmentosum.

Patients with the genetic disease xeroderma pigmentosum (XP) lack the capacity to carry out a specific type of DNA repair process called nucleotide excision repair (NER). The NER pathway plays a critical role in the repair of DNA damage resulting from ultraviolet (UV) radiation. A subset of XP patients develops a profound neurodegenerative condition known as XP neurological disease. Robbins and colleagues [Andrews A, Barrett S, Robbins J (1978) Xeroderma pigmentosum neurological abnormalities correlate with the colony forming ability after ultraviolet irradiation. Proc Natl Acad Sci U S A 75:1984-1988] hypothesized that since UV light cannot reach into the human brain, XP neurological disease results from some form of endogenous DNA damage that is normally repaired by the NER pathway. In the absence of NER, the damage accumulates, causing neuronal death by blocking transcription. In this manuscript, I consider the evidence that a particular class of oxidative DNA lesions, the 8,5'-cyclopurine-2'-deoxynucleosides, fulfills many of the criteria expected of neurodegenerative DNA lesions in XP. Specifically, these lesions are chemically stable, endogenous DNA lesions that are repaired by the NER pathway but not by any other known process, and strongly block transcription by RNA polymerase II in cells from XP patients. A similar set of criteria might be used to evaluate other candidate DNA lesions responsible for neurological diseases resulting from defects in other DNA repair mechanisms as well.

An unusual sugar conformation in the structure of an RNA/DNA decamer of the polypurine tract may affect recognition by RNase H.

Retroviral conversion of single-stranded RNA into double-stranded DNA requires priming for each strand. While host cellular t-RNA serves as primer for the first strand, the viral polypurine tract (PPT) is primer for the second. Therefore, polypurine tracts of retroviruses are essential for viral replication by reverse transcriptase (RT). These purine tracts are resistant to cleavage during first strand synthesis. In obtaining the primer for second strand synthesis, the RNase H function of RT must cleave the PPT exactly for in vivo transcription to proceed efficiently and proper integration to occur. At the RNase H active site the protein makes contacts primarily along the backbone, with hydrogen bonds to the sugar-phosphate oxygen atoms. A high-resolution structure (1.10A) of the first ten base-pairs of the RNA/DNA hybrid PPT, r-(c-a-a-a-g-a-a-a-a-g)/d-(C-T-T-T-T-C-T-T-T-G), contains the highly deformable r-(a-g-a) steps found in retroviral polypurine tracts. This r-(a-g-a) motif is utilized in the "unzipping" or unpairing of bases that occurs when RT binds a malleable PPT. Another unusual feature found in our high-resolution PPT structure is the sugar switch at RNA adenine 2. All the RNA sugars are the expected C3'-endo, except sugar 2, which is C2'-endo, characteristic of B-form sugars. This local A-to-B conversion adversely affects the pattern of hydrogen bonds from protein to sugar-phosphate backbone, disrupting the catalytic site. Disruption could cause the enzyme to pause at the 5'-end of the PPT, leaving it intact. Pyrimidine-purine (YR) steps are most deformable and the T-A step especially can undergo A-to-B transitions readily.

[Phenotypic manifestation of the pnd mutation, which promotes purine nucleoside cleavage by Escherichia coli K-12 cells, in the genome of strains defective in the metabolism of nucleic acid precursors]. / Fenotipicheskoe proiavlenie mutatsii pnd, sposobstvuiushchei rasshchepleniiu purinovykh nukleozidov kletkami Escherichia coli K-12 v genome shtammov, defektnykh po metabolizmu predshestvennikov nukleinovykh kislot.

Strains of Escherichia coli K-12 containing both pnd1 mutation, rendering bacteria capable to catabolize purine nucleosides without participation of purine nucleoside phosphorylase (pup gene), and mutations in several genes of purine metabolism or nucleosides catabolism have been constructed. The introduction of the deletion mutation in adenosine deaminase gene (add) into the pup pnd genome does not affect the ability of mutants to utilize adenosine and deoxyadenosine as the sole carbon and energy sources. Mutations affecting purine phosphoribosyltransferases (hpt and gpt) block the ability of pup pnd mutants to utilize hypoxanthine, guanine and their deoxyribonucleosides and also xanthine and xanthosine as the only purine source. A mutation in deoxyribomutase (drm) disturbs the ability of pnd mutants to use all purine ribo- and deoxy-ribonucleosides as carbon and energy sources, whereas a mutation in deoxyriboaldolase (dra) only disturbs utilization of deoxyribonucleosides. These data seem to indicate that the activity promoted by pnd mutations catalyzes the cell reaction of irreversible phosphorolytic cleavage of the N-glycoside bond of the purine nucleosides molecules: purine nucleoside + phosphate leads to purine + pentose-1-phosphate. It is suggested that pnd mutations affect the structural gene of some phosphorolytic enzyme and modify its substrate specificity. Evidence is presented that the structural gene of a new nucleoside phosphorylase is not sensitive to catabolite repression.