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.

Reaction of Electrons with DNA: Radiation Damage to Radiosensitization.

This review article provides a concise overview of electron involvement in DNA radiation damage. The review begins with the various states of radiation-produced electrons: Secondary electrons (SE), low energy electrons (LEE), electrons at near zero kinetic energy in water (quasi-free electrons, (e-qf)) electrons in the process of solvation in water (presolvated electrons, e-pre), and fully solvated electrons (e-aq). A current summary of the structure of e-aq, and its reactions with DNA-model systems is presented. Theoretical works on reduction potentials of DNA-bases were found to be in agreement with experiments. This review points out the proposed role of LEE-induced frank DNA-strand breaks in ion-beam irradiated DNA. The final section presents radiation-produced electron-mediated site-specific formation of oxidative neutral aminyl radicals from azidonucleosides and the evidence of radiosensitization provided by these aminyl radicals in azidonucleoside-incorporated breast cancer cells.

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin.

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics.IMPORTANCE POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments.

Genetic modification of large DNA fragments (gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity. In this study, we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction (PCR) targeting combined with Gibson assembly. In this strategy, the biosynthetic genes for peptidyl moieties (HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid (CPOAA) from the polyoxin biosynthetic gene cluster to generate a ~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette. The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic, polynik A, was obtained and verified. This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.

DNA and RNA Pyrimidine Nucleobase Alkylation at the Carbon-5 Position.

The carbon 5 of pyrimidine nucleobases is a privileged position in terms of nucleoside modification in both DNA and RNA. The simplest modification of uridine at this position is methylation leading to thymine. Thymine is an integral part of the standard nucleobase repertoire of DNA that is synthesized at the nucleotide level. However, it also occurs in RNA, where it is synthesized posttranscriptionally at the polynucleotide level. The cytidine analogue 5-methylcytidine also occurs in both DNA and RNA, but is introduced at the polynucleotide level in both cases. The same applies to a plethora of additional derivatives found in nature, resulting either from a direct modification of the 5-position by electrophiles or by further derivatization of the 5-methylpyrimidines. Here, we review the structural diversity of these modified bases, the variety of cofactors that serve as carbon donors, and the common principles shared by enzymatic mechanisms generating them.

Metabolic engineering of an industrial polyoxin producer for the targeted overproduction of designer nucleoside antibiotics.

Polyoxin and nikkomycin are naturally occurring peptidyl nucleoside antibiotics with potent antifungal bioactivity. Both exhibit similar structural features, having a nucleoside skeleton and one or two peptidyl moieties. Combining the refactoring of the polyoxin producer Streptomyces aureochromogenes with import of the hydroxypyridylhomothreonine pathway of nikkomycin allows the targeted production of three designer nucleoside antibiotics designated as nikkoxin E, F, and G. These structures were determined by NMR and/or high resolution mass spectrometry. Remarkably, the introduction of an extra copy of the nikS gene encoding an ATP-dependent ligase significantly enhanced the production of the designer antibiotics. Moreover, all three nikkoxins displayed improved bioactivity against several pathogenic fungi as compared with the naturally-occurring antibiotics. These data provide a feasible model for high efficiency generation of nucleoside antibiotics related to polyoxins and nikkomycins in a polyoxin cell factory via synthetic biology strategy.

GouR, a TetR family transcriptional regulator, coordinates the biosynthesis and export of gougerotin in Streptomyces graminearus.

Gougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities. gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed that gouR plays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of the gouL-to-gouB operon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions of gouL, gouM, and gouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export in Streptomyces graminearus.

Convergent evolution of AUA decoding in bacteria and archaea.

Deciphering AUA codons is a difficult task for organisms, because AUA and AUG specify isoleucine (Ile) and methionine (Met), separately. Each of the other purine-ending sense co-don sets (NNR) specifies a single amino acid in the universal genetic code. In bacteria and archaea, the cytidine derivatives, 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm(2)C or agmatidine), respectively, are found at the first letter of the anticodon of tRNA(Ile) responsible for AUA codons. These modifications prevent base pairing with G of the third letter of AUG codon, and enable tRNA(Ile) to decipher AUA codon specifically. In addition, these modifications confer a charging ability of tRNA(Ile) with Ile. Despite their similar chemical structures, L and agm(2)C are synthesized by distinctive mechanisms and catalyzed by different classes of enzymes, implying that the analogous decoding systems for AUA codons were established by convergent evolution after the phylogenic split between bacteria and archaea-eukaryotes lineages following divergence from the last universal common ancestor (LUCA).

Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the "pathway redundancy" in metabolic networks.

BACKGROUND: Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the "pathway redundancy" of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer. RESULTS: Here we report the genetic characterization of CPOAA biosynthesis with revealing a "pathway redundancy" in metabolic networks. Independent mutation of the four genes (polL-N and polP) directly resulted in the accumulation of polyoxin I, suggesting their positive roles for CPOAA biosynthesis. Moreover, the individual mutant of polN and polP also partially retains polyoxin production, suggesting the existence of the alternative homologs substituting their functional roles. CONCLUSIONS: It is unveiled that argA and argB in L-arginine biosynthetic pathway contributed to the "pathway redundancy", more interestingly, argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide an example for the research on the "pathway redundancy" in metabolic networks, and lay a solid foundation for targeted enhancement of polyoxin production with synthetic biology strategies.

Cloning, heterologous expression, and characterization of the gene cluster required for gougerotin biosynthesis.

Gougerotin, a peptidyl nucleoside antibiotic, possesses antitumor, antiviral, antibacterial, antimycoplasma, anthelmintic, and acaricidal activities. Here, we report the cloning of a complete gougerotin biosynthetic gene cluster from Streptomyces graminearus and heterologous production of gougerotin in Streptomyces coelicolor. Sequence analysis of a 28.7 kb DNA fragment indicated that the cluster consists of 25 open reading frames (ORFs). Gene disruption and genetic complementation experiments revealed that 15 of the 25 ORFs are required for gougerotin biosynthesis. A gougerotin biosynthetic pathway was proposed based on the analyses of bioinformatics and intermediates accumulated in selected gene inactivation mutants. These studies substantially promoted our understanding of gougerotin biosynthesis and provide "building blocks" for combinatorial biosynthesis using genes encoding different enzymes in nucleoside antibiotics.

Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics.

Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer.

Life without the essential bacterial tRNA Ile2-lysidine synthetase TilS: a case of tRNA gene recruitment in Bacillus subtilis.

In eubacteria, the post-transcriptional modification of the wobble cytidine of the CAU anticodon in a precursor tRNA(Ile2) to a lysidine residue (2-lysyl-cytidine, abbreviated as L) allows the amino acid specificity to change from methionine to isoleucine and the codon decoding specificity to shift from AUG to AUA. The tilS gene encoding the enzyme that catalyses this modification is widely distributed. However, some microbial species lack a tilS gene, indicating that an alternative strategy exists to accurately translate the AUA codon into Ile. To determine whether a TilS-dependent bacterium, such as Bacillus subtilis, can overcome the absence of lysidine in its tRNA(Ile2) (CAU), we analysed the suppressor mutants of a tilS-thermosensitive allele. These tilS-suppressor mutants carry a substitution of the wobble guanosine into thymidine in one of the tRNA(Ile1) genes (the original GAT anticodon is changed to a TAT). In absence of TilS activity, the AUA codons are translated into isoleucine by the suppressor tRNA(Ile1), although a low level of AUA codons is also mistranslated into methionine. Results are in agreement with rare cases of eubacteria (and archaea), which naturally lack the tilS gene (or tiaS in archaea) but contain a tRNA(Ile2) gene containing a TAT instead of a CAT anticodon.

Hybrid antibiotics with the nikkomycin nucleoside and polyoxin peptidyl moieties.

Acting as competitive inhibitors of chitin synthase, nikkomycins and polyoxins are potent antibiotics against pathogenic fungi. Taking advantage of the structural similarities between these two peptidyl nucleoside antibiotics, genes required for the biosynthesis of the dipeptidyl moiety of polyoxin from Streptomyces cacaoi were introduced into a Streptomyces ansochromogenes mutant producing the nucleoside moiety of nikkomycin X. Two hybrid antibiotics were generated. One of them was identified as polyoxin N, and the other, a novel compound, was named polynik A. The hybrid antibiotics exhibited merits from both parents: they had better inhibitory activity against phytopathogenic fungi than polyoxin B, and were more stable under different pH and temperature conditions than nikkomycin X. This study demonstrates the use of the combinatorial biosynthetic approach to produce valuable and novel hybrid antibiotics with improved properties.

Enhancement of the diversity of polyoxins by a thymine-7-hydroxylase homolog outside the polyoxin biosynthesis gene cluster.

Polyoxins consist of 14 structurally variable components which differentiate at three branch sites of the carbon skeleton. Open reading frame (ORF) SAV_4805 of Streptomyces avermitilis, showing similarity to thymine-7-hydroxylase, was proved to enhance the diversity of polyoxins at the C-5 site of the 1-(5'-amino-5'-deoxy-ß-d-allofuranuronosyl) pyrimidine moiety.

Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics.

Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics that act on the translocase MraY to block bacterial cell wall assembly. To elucidate the biosynthetic logic of pacidamcyins, a putative gene cluster was identified by 454 shotgun genome sequencing of the producer Streptomyces coeruleorubidus NRRL 18370. The 31-kb gene cluster encodes 22 proteins (PacA-V), including highly dissociated nonribosomal peptide synthetase (NRPS) modules and a variety of tailoring enzymes. Gene deletions confirmed that two NRPSs, PacP and PacO, are required for the biosynthesis of pacidamycins. Heterologous expression and in vitro assays of PacL, PacO, and PacP established reversible formation of m-Tyr-AMP, l-Ala-AMP, and diaminopropionyl-AMP, respectively, consistent with the amino acids found in pacidamycin scaffolds. The unusual Ala(4)-Phe(5) dipeptidyl ureido linkage was formed during in vitro assays containing purified PacL, PacJ, PacN, and PacO. Both the genetic and enzymatic studies validate identification of the biosynthetic genes for this subclass of uridyl peptide antibiotics and provide the basis for future mechanistic study of their biosynthesis.

Pacidamycin biosynthesis: identification and heterologous expression of the first uridyl peptide antibiotic gene cluster.

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3'-deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.

PolY, a transcriptional regulator with ATPase activity, directly activates transcription of polR in polyoxin biosynthesis in Streptomyces cacaoi.

polY, a transcriptional regulatory gene in the polyoxin biosynthetic cluster of Streptomyces cacaoi, was analysed, and its deduced product (PolY) showed amino acid sequence homology to AfsR from Streptomyces coelicolor A3(2). PolY contains an OmpR-like DNA binding domain at its N-terminal and an ATPase domain in the middle of the protein. Disruption of polY abolished polyoxin biosynthesis, which could be restored by the integration of a single copy of polY into the chromosome of the disruption mutant. Transcription of polR, a pathway-specific regulatory gene of polyoxin biosynthesis, was controlled by polY. Electrophoretic mobility shift assay and DNase I protection experiments indicated that PolY bound to the promoter region of polR, and the binding site contained a direct nucleotide repeat typical of Streptomyces antibiotic regulatory protein binding sites. PolY exhibited ATPase activity in vitro. Additionally, binding of ADP/ATPgammaS to ATPase domain triggered the oligomerization of PolY and enhanced its DNA binding activity. Consistently, further experiments in vivo demonstrated that changes of ADP/ATP concentrations significantly affected PolY activity in the cell. These results suggested that the ATPase domain might be a sensor of endogenous pool of ADP/ATP, whose change modulated PolY activity under the physiological conditions.

Characterization of the polyoxin biosynthetic gene cluster from Streptomyces cacaoi and engineered production of polyoxin H.

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics.

Hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency: a biochemical, genetic and clinical overview.

Pyrimidine 5' -nucleotidase (P5'N-1) deficiency is the third most common enzyme abnormality after glucose 6-phosphate dehydrogenase and pyruvate kinase causing hereditary non-spherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait. The degree of hemolysis is generally mild-to moderate. The hallmark of this enzyme deficiency is the presence of pronounced basophylic stippling in red blood cell peripheral blood smear together with accumulation of pyrimidine nucleotides within erythrocytes. No correlation has been found between residual activity and degree of hemolysis. The structural human gene for P5'N-1 is now available and fifteen different mutations had been identified so far. More recently, a functional analysis of P5'N-1 mutants had been performed providing a rationale for the pathological effects of the mutations. All mutations investigated affect amino acid residues unambiguously essential for the catalytic efficiency and/or protein stability, suggesting drastic reduction of the enzyme activity in red blood cells of patients affected by the disorder. Nevertheless, some patients exhibit high residual P5'N-1 activity, suggesting that P5'N-1 deficiency is compensate by other nucleotidases and/or alternative pathways in nucleotide metabolism. No specific therapy for P5'N-1 deficiency is now available.