Nucleoside Biosynthesis Research Articles

Inhibiting pyrimidine biosynthesis impairs Peste des Petits Ruminants Virus replication through depletion of nucleoside pools and activation of cellular immunity

Replication of peste des petits ruminants virus (PPRV) strongly depends on the cellular environment and resources of host cells including nucleoside pool. Thus, enzymes involved in nucleoside biosynthesis (such as pyrimidine biosynthesis pathway) are regarded as attractive targets for antiviral drug development. Here, we demonstrate that brequinar (BQR) and leflunomide (LFM) which are two specific inhibitors of DHODH enzyme and 6-azauracil (6-AU) which is an ODase enzyme inhibitor robustly inhibit PPRV replication in HEK293T cell line as well as in peripheral blood mononuclear cells isolated from goat. We further demonstrate that these agents exert anti-PPRV activity via the depletion of purimidine nucleotide. Interestingly, these inhibitors can trigger the transcription of antiviral interferon-stimulated genes (ISGs). However, the induction of ISGs is largely independent of the classical JAK-STAT pathway. Combination of BQR with interferons (IFNs) exerts enhanced ISG induction and anti-PPRV activity. Taken together, this study reveals an unconventional novel mechanism of crosstalk between nucleotide biosynthesis pathways and cellular antiviral immunity in inhibiting PPRV replication. In conclusion, targeting pyrimidine biosynthesis represents a potential strategy for developing antiviral strategies against PPRV.

Conserved Mechanism of 2'-Phosphorylation-Aided Amide Ligation in Peptidyl Nucleoside Biosynthesis.

Peptidyl nucleoside antifungals, represented by nikkomycins and polyoxins, consist of an unusual six-carbon nucleoside [aminohexuronic acid (AHA)] ligated to a nonproteinogenic amino acid via an amide bond. A recent study suggested that AHA is biosynthesized through cryptic phosphorylation, where a 2'-phosphate is introduced early in the pathway and required to form AHA. However, whether 2'-phosphorylation is necessary for the last step of biosynthesis, the formation of the amide bond between AHA and nonproteinogenic amino acids, remains ambiguous. Here, we address this question with comprehensive in vitro and in vivo characterizations of PolG and NikS, which together provide strong evidence that amide ligation proceeds with 2'-phosphorylated substrates in both pathways. Our results suggest that 2'-phosphorylation is retained for the entirety of both nikkomycin and polyoxin biosynthesis, providing important insights into how cryptic phosphorylation assists with nucleoside natural product biosynthesis.

Cryptic phosphorylation in nucleoside natural product biosynthesis.

Kinases are annotated in many nucleoside biosynthetic gene clusters (BGCs) but generally are considered responsible only for self-resistance. Here, we report an unexpected 2’-phosphorylation of nucleoside biosynthetic intermediates in the nikkomycin and polyoxin pathways. This phosphorylation is a unique cryptic modification as it is introduced in the third of seven steps during aminohexuronic acid (AHA) nucleoside biosynthesis, retained throughout the pathway’s duration, and is removed in the last step of the pathway. Bioinformatic analysis of reported nucleoside BGCs suggests the presence of cryptic phosphorylation in other pathways and the importance of functional characterization of kinases in nucleoside biosynthetic pathways in general. This study also functionally characterized all of the enzymes responsible for AHA biosynthesis and revealed that AHA is constructed via a unique oxidative C-C bond cleavage reaction. The results suggest a divergent biosynthetic mechanism for three classes of antifungal nucleoside natural products.

Genetic Polymorphism of ADORA2A Is Associated With the Risk of Epilepsy and Predisposition to Neurologic Comorbidity in Chinese Southern Children.

Epilepsy, a common disorder of the brain, exhibits a high morbidity rate in children. Childhood epilepsy (CE) is frequently comorbid with neurologic and developmental disorders, sharing underlying genetic factors. This study aimed to investigate the impact of ADORA2A, BDNF, and NTRK2 gene polymorphisms on the risk of childhood epilepsy and their associations with predisposition to epileptic comorbidities. A total of 444 children were enrolled in this study, and three single nucleotide polymorphisms, including ADORA2A rs2298383, BDNF rs6265, and NTRK2 rs1778929, were genotyped. The frequency distribution of genotypes was compared not only between CE patients and healthy children but also between CE patients with and without comorbidities. The results indicated that the carriers of ADORA2A rs2298383 TT genotype tended to have a lower risk of epilepsy (OR = 0.48, 95% CI = 0.30–0.76), while the CT genotype was related to a higher risk (OR = 1.56, 95% CI = 1.06–2.27). The ADORA2A rs2298383 CC genotype predisposed CE patients to comorbid neurologic disorders (OR = 2.76, 95% CI = 1.31–5.80). Genetic variations in BDNF rs6265 and NTRK2 rs1778929 had no significant association with CE and its comorbidities. Fourteen ADORA2A target genes related to epilepsy were identified by the protein–protein interaction analysis, which were mainly involved in the biological processes of “negative regulation of neuron death” and “purine nucleoside biosynthetic process” through the gene functional enrichment analysis. Our study revealed that the genetic polymorphism of ADORA2A rs2298383 was associated with CE risk and predisposition to neurologic comorbidity in children with epilepsy, and the involved mechanism might be related to the regulation of neuron death and purine nucleoside biosynthesis.

The Gut Microbiome Is Associated with Clinical Response to Anti-PD-1/PD-L1 Immunotherapy in Gastrointestinal Cancer.

We report on a comprehensive analysis of the gut microbiomes of patients with gastrointestinal (GI) cancer receiving anti-PD-1/PD-L1 treatment. The human gut microbiota has been associated with clinical responses to anti-PD-1/PD-L1 immunotherapy in melanoma, non-small cell lung cancer, and renal cell carcinoma. We aimed to investigate this association in GI cancers. We also identified bacterial taxa with patient stratification potential. We recruited 74 patients with advanced-stage GI cancer receiving anti-PD-1/PD-L1 treatment and collected their fecal samples prior to and during immunotherapy, along with clinical evaluations. Our 16S rRNA taxonomy survey on the fecal samples revealed an elevation of the Prevotella/Bacteroides ratio in patients, with a preferred response to anti-PD-1/PD-L1 treatment, and a particular subgroup of responders harboring a significantly higher abundance of Prevotella, Ruminococcaceae, and Lachnospiraceae The shotgun metagenomes of the same samples showed that patients exhibiting different responses had differential abundance of pathways related to nucleoside and nucleotide biosynthesis, lipid biosynthesis, sugar metabolism, and fermentation to short-chain fatty acids (SCFA). Gut bacteria that were capable of SCFA production, including Eubacterium, Lactobacillus, and Streptococcus, were positively associated with anti-PD-1/PD-L1 response across different GI cancer types. We further demonstrated that the identified bacterial taxa were predictive of patient stratification in both our cohort and melanoma patients from two previously published studies. Our results thus highlight the impact of gut microbiomes on anti-PD-1/PD-L1 outcomes, at least in a subset of patients with GI cancer, and suggest the potential of the microbiome as a marker for immune-checkpoint blockade responses.See articles by Tomita et al., p. 1236, and Hakozaki et al., p. 1243.

Abstract 555: Mitochondrial fusion exerts KRAS-dependent therapeutic synergy with gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is driven by activating Kras mutations which cause a fragmented mitochondrial phenotype. We recently demonstrated that leflunomide, an FDA-approved drug for rheumatoid arthritis, could be repurposed for pancreatic cancer therapy. The mode of action is through the activation of mitochondrial fusion, which inhibits PDAC growth in a KRAS-dependent fashion. Leflunomide also has the off-target effect of inhibiting dihydroorotate dehydrogenase (DHODH), which is critical for pyrimidine biosynthesis. Gemcitabine is part of the standard of care for pancreatic cancer and also inhibits nucleoside biosynthesis. Accordingly, we hypothesized that leflunomide might synergize with gemcitabine chemotherapy to further improve outcomes. In this pre-clinical study, we attempt to provide proof-of-concept for combining leflunomide with gemcitabine/nab-paclitaxel, the current gold standard for PDAC, in order to characterize a synergistic effect and to provide evidence for potentially repurposing the drug for clinical use. Methods: We heterotopically implanted patient-derived pancreatic cancer cell lines with an activating KRAS mutations (MDA-PATC148) or WT KRAS (MDA-PATC153) into the flanks of immunocompromised (NOD-SCID) mice. The tumors were grown to 5mm in size then assigned to one of four treatment groups: (1) vehicle control, (2) gemcitabine/nab-paclitaxel only, and (3) leflunomide + gemcitabine/nab-paclitaxel. Leflunomide was dosed at 20 mg/kg and given concurrently with gemcitabine and nab-paclitaxel. Gemcitabine was administered IP 2x/week at 100 mg/kg and nab-paclitaxel given once per week by tail vein at 10 mg/kg. Results: The combination of leflunomide with gemcitabine/nab-paclitaxel synergistically impeded tumor proliferation and reduced the median tumor volume by more than 50% when compared to vehicle (102.6 vs. 294.8 mm3, ****P-value < 0.0001 by Mann-Whitney U test) or gemcitabine/nab-paclitaxel only group (102.6 mm3 vs. 211.7 mm3, **P-value < 0.01 by Mann-Whitney U test) in KRAS mutant PATC148 mice. Interestingly, the addition of leflunomide to gemcitabine/nab-paclitaxel did not further improve tumor responses in KRAS wild-type PATC153 tumors, suggesting a dependence on mutant KRAS for therapeutic efficacy of the repurposed mitochondrial fusion actiator, leflunomide. Conclusions: Leflunomide exhibits therapeutic synergy with standard-of-care gemcitabine/nab-paclitaxel in patient-derived PDAC lines that harbor KRAS mutations, but are ineffective in KRAS WT lines, suggesting that this combination might be effective not only in PDAC, but in other cancer with mutant KRAS, such as cancers of colon, lung, or bile ducts. Citation Format: Nicholas D. Nguyen, Meifang Yu, Tara N. Fujimoto, Abagail Delahoussaye, Yanqing Huang, Manuel A. Estrada, Cullen M. Taniguchi. Mitochondrial fusion exerts KRAS-dependent therapeutic synergy with gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 555.

Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis

Several nucleoside antibiotics are structurally characterized by a 5′′-amino-5′′-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside, (5′S,6′S)-5′-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origins of which have yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-l-methionine (AdoMet) as the direct precursor of the N-alkylamine, but unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5′-phophosate (PLP)-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to utilizing a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases.

Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to heterocyclic bases, giving α-d-ribose-1-phosphate or α-d-2-deoxyribose-1-phosphate. These enzymes are involved in salvage pathways of nucleoside biosynthesis. The level of these enzymes is often elevated in tumors, which can be used as a marker for cancer diagnosis. This review presents the analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases of the first (NP-1) family, which includes hexameric and trimeric purine nucleoside phosphorylases (EC 2.4.2.1), hexameric and trimeric 5′-deoxy-5′-methylthioadenosine phosphorylases (EC 2.4.2.28), and uridine phosphorylases (EC 2.4.2.3). Nucleosides adopt similar conformations in complexes, with these conformations being significantly different from those of free nucleosides. In complexes, pentofuranose rings of all nucleosides are at the W region of the pseudorotation cycle that corresponds to the energy barrier to the N↔S interconversion. In most of the complexes, the orientation of the bases with respect to the ribose is in the high-syn region in the immediate vicinity of the barrier to syn ↔ anti transitions. Such conformations of nucleosides in complexes are unfavorable when compared to free nucleosides and they are stabilized by interactions with the enzyme. The sulfate (or phosphate) ion in the active site of the complexes influences the conformation of the furanose ring. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme–substrate complex formation for this enzyme group.

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli.

The nitrile reductase QueF catalyzes NADPH‐dependent reduction of the nitrile group of preQ0 (7‐cyano‐7‐deazaguanine) into the primary amine of preQ1 (7‐aminomethyl‐7‐deazaguanine), a biologically unique reaction important in bacterial nucleoside biosynthesis. Here we have discovered that the QueF from Escherichia coli—its D197A and E89L variants in particular (apparent k cat≈10−2 min−1)—also catalyze the slow hydration of the C5=C6 double bond of the dihydronicotinamide moiety of NADPH. The enzymatically C6‐hydrated NADPH is a 3.5:1 mixture of R and S forms and rearranges spontaneously through anomeric epimerization (β→α) and cyclization at the tetrahydronicotinamide C6 and the ribosyl O2. NADH and 1‐methyl‐ or 1‐benzyl‐1,4‐dihydronicotinamide are not substrates of the enzymatic hydration. Mutagenesis results support a QueF hydratase mechanism, in which Cys190—the essential catalytic nucleophile for nitrile reduction—acts as the general acid for protonation at the dihydronicotinamide C5 of NADPH. Thus, the NADPH hydration in the presence of QueF bears mechanistic resemblance to the C=C double bond hydration in natural hydratases.

Transcriptomic Behavior of Salmonella enterica Newport in Response to Oxidative Sanitizers

Agricultural water is a known source of contamination to fresh produce and can contain foodborne pathogens including Salmonella enterica, pathogenic Escherichia coli, Listeria monocytogenes, and Campylobacter jejuni. To mitigate such risks, antimicrobial agents such as hypochlorites and peroxyacetic acid (PAA) can be applied to in-line irrigation systems as well as to water used in postharvest washing. Although these compounds are effective and widely used, some pathogenic bacteria adapt to survive exposure. RNA sequencing was used to analyze the Salmonella Newport transcriptome after exposure to sodium hypochlorite (NaOCl) and PAA in a simulated agricultural water system. Overall cellular adaptive response was determined quantitatively as a function of overall gene expression of the >4,000 genes in the Salmonella Newport genome. Differentially expressed genes ranged from 11 due to 10-ppm NaOCl treatment, 316 due to 20-ppm NaOCl treatment, 1,719 due to 10-ppm PAA treatment, and 2,010 due to 20-ppm PAA treatment compared with that of the controls (water only). Differentially expressed transcripts included cellular functions such as biosynthesis, degradation, energy generation, and nonmetabolically linked functions. Oxidative exposure upregulated genes associated with key virulence, attachment, and gene transfer. Amino acid biosynthesis was upregulated due to NaOCl exposure but primarily downregulated when Salmonella Newport was exposed to PAA. Slight upregulation occurred in nucleoside and nucleotide biosynthesis, a known DNA repair mechanism seen during exposure to sanitizers. Our results indicate that Salmonella Newport reacts differently when exposed to NaOCl versus PAA, despite oxidative activity being the primary modes of antimicrobial action of both compounds.

A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5′-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5′-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP.

Comparative analysis of differential proteome-wide protein-protein interaction network of Methanobrevibacter ruminantium M1

A proteome-wide protein-protein interaction (PPI) network of Methanobrevibacter ruminantium M1 (MRU), a predominant rumen methanogen, was constructed from its metabolic genes using a gene neighborhood algorithm and then compared with closely related rumen methanogens Using proteome-wide PPI approach, we constructed network encompassed 2194 edges and 637 nodes interacting with 634 genes. Network quality and robustness of functional modules were assessed with gene ontology terms. A structure-function-metabolism mapping for each protein has been carried out with efforts to extract experimental PPI concomitant information from the literature. The results of our study revealed that some topological properties of its network were robust for sharing homologous protein interactions across heterotrophic and hydrogenotrophic methanogens. MRU proteome has shown to establish many PPI sub-networks for associated metabolic subsystems required to survive in the rumen environment. MRU genome found to share interacting proteins from its PPI network involved in specific metabolic subsystems distinct to heterotrophic and hydrogenotrophic methanogens. Across these proteomes, the interacting proteins from differential PPI networks were shared in common for the biosynthesis of amino acids, nucleosides, and nucleotides and energy metabolism in which more fractions of protein pairs shared with Methanosarcina acetivorans. Our comparative study expedites our knowledge to understand a complex proteome network associated with typical metabolic subsystems of MRU and to improve its genome-scale reconstruction in the future.

Changes in metabolic profiling of sugarcane leaves induced by endophytic diazotrophic bacteria and humic acids.

Plant growth-promoting bacteria (PGPB) and humic acids (HA) have been used as biostimulants in field conditions. The complete genomic and proteomic transcription of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus is available but interpreting and utilizing this information in the field to increase crop performance is challenging. The identification and characterization of metabolites that are induced by genomic changes may be used to improve plant responses to inoculation. The objective of this study was to describe changes in sugarcane metabolic profile that occur when HA and PGPB are used as biostimulants. Inoculum was applied to soil containing 45-day old sugarcane stalks. One week after inoculation, the methanolic extracts from leaves were obtained and analyzed by gas chromatography coupled to time-of-flight mass spectrometry; a total of 1,880 compounds were observed and 280 were identified in all samples. The application of HA significantly decreased the concentration of 15 metabolites, which generally included amino acids. HA increased the levels of 40 compounds, and these included metabolites linked to the stress response (shikimic, caffeic, hydroxycinnamic acids, putrescine, behenic acid, quinoline xylulose, galactose, lactose proline, oxyproline and valeric acid) and cellular growth (adenine and adenosine derivatives, ribose, ribonic acid and citric acid). Similarly, PGPB enhanced the level of metabolites identified in HA-treated soils; e.g., 48 metabolites were elevated and included amino acids, nucleic acids, organic acids, and lipids. Co-inoculation (HA+PGPB) boosted the level of 110 metabolites with respect to non-inoculated controls; these included amino acids, lipids and nitrogenous compounds. Changes in the metabolic profile induced by HA+PGPB influenced both glucose and pentose pathways and resulted in the accumulation of heptuloses and riboses, which are substrates in the nucleoside biosynthesis and shikimic acid pathways. The mevalonate pathway was also activated, thus increasing phytosterol synthesis. The improvement in cellular metabolism observed with PGPB+HA was compatible with high levels of vitamins. Glucuronate and amino sugars were stimulated in addition to the products and intermediary compounds of tricarboxylic acid metabolism. Lipids and amino acids were the main compounds induced by co-inoculation in addition to antioxidants, stress-related metabolites, and compounds involved in cellular redox. The primary compounds observed in each treatment were identified, and the effect of co-inoculation (HA+PGPB) on metabolite levels was discussed.

Periodic fasting starves cisplatin-resistant cancers to death.

Treatment of cancers with the cytotoxic agent cisplatin frequently evokes resistance, accompanied by rewiring of metabolic pathways, limiting its clinical use. Recent research by Obrist et al (2018) shows that cisplatin‐resistant growth of lung adenocarcinoma is particularly vulnerable to periodic fasting cycles and starvation‐induced cell death, due to its dependency on glutamine, required for nucleoside biosynthesis, suggesting an opportunity for nutritional anti‐cancer interventions.

Spatially Restricting Bioorthogonal Nucleoside Biosynthesis Enables Selective Metabolic Labeling of the Mitochondrial Transcriptome.

The cellular RNA pool in animals arises from two separate genomes stored in the nucleus and multiple mitochondria. Chemical methods to track nascent RNA synthesis are unable to distinguish between these two with stringency. Herein, we report that spatially restricting bioorthogonal nucleoside biosynthesis enables, for the first time, selective metabolic labeling of the RNA transcribed in the mitochondria. We envision that this approach could open the door for heretofore-impossible analyses of mitochondrial RNA. Beyond our results revealed herein, our approach provides a roadmap for researchers to begin to design strategies to examine biomolecules within subcellular compartments.

Quantitative Proteomics of Strong and Weak Biofilm Formers of Enterococcus faecalis Reveals Novel Regulators of Biofilm Formation

Enterococcus faecalis is a bacterial pathogen associated with both endodontic and systemic infections. The biofilm formation ability of E. faecalis plays a key role in its virulence and drug resistance attributes. The formation of E. faecalis biofilms on implanted medical devices often results in treatment failure. In the present study, we report protein markers associated with the biofilm formation ability of E. faecalis using iTRAQ-based quantitative proteomics approach. In order to elucidate the biofilm-associated protein markers, we investigated the proteome of strong and weak biofilm-forming E. faecalis clinical isolates in comparison with standard American Type Culture Collection (ATCC) control strains. Comparison of E. faecalis strong and weak biofilm-forming clinical isolates with ATCC control strains showed that proteins associated with shikimate kinase pathway and sulfate transport were up-regulated in the strong biofilm former, while proteins associated with secondary metabolites, cofactor biosynthesis, and tetrahydrofolate biosynthesis were down-regulated. In the weak biofilm former, proteins associated with nucleoside and nucleotide biosynthesis were up-regulated, whereas proteins associated with sulfate and sugar transport were down-regulated. Further pathway and gene ontology analyses revealed that the major differences in biofilm formation arise from differences in metabolic activity levels of the strong and weak biofilm formers, with higher levels of metabolic activity observed in the weak biofilm former. The differences in metabolic activity could therefore be a major determinant of the biofilm ability of E. faecalis The new markers identified from this study can be further characterized in order to understand their exact role in E. faecalis biofilm formation ability. This, in turn, can lead to numerous therapeutic benefits in the treatment of this oral and systemic pathogen. The data has been deposited to the ProteomeXchange with identifier PXD006542.

Development of a high efficient biocatalyst by oriented covalent immobilization of a novel recombinant 2′-N-deoxyribosyltransferase from Lactobacillus animalis

The 2′-N-deoxyribosyltransferases [NDT; EC 2.4.2.6] are a group of enzymes widely used as biocatalysts for nucleoside biosynthesis. In this work, the molecular cloning, expression and purification of a novel NDT from Lactobacillus animalis (LaNDT) have been reported. On the other hand, biocatalyst stability has been significantly enhanced by multipoint covalent immobilization using a hetero-functional support activated with nickel-chelates and glyoxyl groups. The immobilized enzyme could be reused for more than 300 h and stored during almost 3 months without activity loss. Besides, the obtained derivative (Ni2+-Gx-LaNDT) was able to biosynthesize 88 mg floxuridine/g biocatalyst after 1 h of reaction. In this work, a green bioprocess by employing an environmentally friendly methodology was developed, which allowed the obtaining of a compound with proven anti-tumor activity. Therefore, the obtained enzymatic biocatalyst meets the requirements of high activity, stability, and short reaction times needed for low-cost production in a future preparative application.

Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coli IscS and Human NFS1.

The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron-sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human l-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of ∼60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm5s2U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present.

Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond.

Infections with flaviviruses, such as dengue, West Nile virus and the recently re-emerging Zika virus, are an increasing and probably lasting global risk. This Review summarizes and comments on the opportunities for broad-spectrum agents that are active against multiple flaviviruses. Broad-spectrum activity is particularly desirable to prepare for the next flaviviral epidemic, which could emerge from as-yet unknown or neglected viruses. Potential molecular targets for broad-spectrum antiflaviviral compounds include viral proteins, such as the viral protease or polymerase, and host targets that are exploited by these viruses during entry and replication, including α-glucosidase and proteins involved in nucleoside biosynthesis. Numerous compounds with broad-spectrum antiviral activity have already been identified by target-specific or phenotypic assays. For other compounds, broad-spectrum activity can be anticipated because of their mode of action and molecular targets.

Enhancement of Nucleoside Production in Hirsutella sinensis Based on Biosynthetic Pathway Analysis.

To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis.