Microbial 5′-nucleotidases: their characteristics, roles in cellular metabolism, and possible practical applications

Abstract5′-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5′-ribonucleotides and 5′-deoxyribonucleotides to their respective nucleosides and phosphate. Most 5′-nucleotidases have broad substrate specificity and are multifunctional enzymes capable of cleaving phosphorus from not only mononucleotide phosphate molecules but also a variety of other phosphorylated metabolites. 5′-Nucleotidases are widely distributed throughout all kingdoms of life and found in different cellular locations. The well-studied vertebrate 5′-nucleotidases play an important role in cellular metabolism. These enzymes are involved in purine and pyrimidine salvage pathways, nucleic acid repair, cell-to-cell communication, signal transduction, control of the ribo- and deoxyribonucleotide pools, etc. Although the first evidence of microbial 5′-nucleotidases was obtained almost 60 years ago, active studies of genetic control and the functions of microbial 5′-nucleotidases started relatively recently. The present review summarizes the current knowledge about microbial 5′-nucleotidases with a focus on their diversity, cellular localizations, molecular structures, mechanisms of catalysis, physiological roles, and activity regulation and approaches to identify new 5′-nucleotidases. The possible applications of these enzymes in biotechnology are also discussed.Key points• Microbial 5′-nucleotidases differ in molecular structure, hydrolytic mechanism, and cellular localization.• 5′-Nucleotidases play important and multifaceted roles in microbial cells.• Microbial 5′-nucleotidases have wide range of practical applications. Graphical abstract

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

Non-metabolic role of UCK2 links EGFR-AKT pathway activation to metastasis enhancement in hepatocellular carcinoma

Up-regulation of Uridine-cytidine kinase 2 (UCK2), a rate-limiting enzyme of the pyrimidine salvage pathway, has been suggested in HCC, but the detailed molecular mechanisms and therapic role of UCK2 remain elusive. Bioinformatic analyses revealed that UCK2 might be a key up-regulated metabolic gene in HCCs. The expressional pattern and prognostic value of UCK2 were further examined in a large number of clinical samples. Functional assays based on site-directed mutagenesis showed that UCK2 promoted cell proliferation in a metabolic manner, but non-catalytically facilitates HCC metastasis. Mechanistically, in response to EGF, UCK2 interacted with EGFR to block EGF-induced EGFR ubiquitination and degradation, which resulted in elevated EGFR-AKT pathway activation and metastasis enhancement in HCCs. Concurrent pharmacological targeting on UCK2 and EGFR showed synergistic effects on HCC treatment. This study disclosed the non-metabolic role of UCK2 and suggested the therapeutic potential of concurrent blocking the metabolic and non-metabolic roles of UCK2 in HCC treatment.

The Synergism Between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia

BackgroundDihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition was recently found to induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors were previously investigated in solid tumors, where they showed promising antiproliferative activity, both in vitro and in vivo. However, their effectiveness was not confirmed in clinical trials, probably due to the pyrimidine salvage pathway that cancer cells could exploit to survive. In this study we investigated the pro-apoptotic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we challenged our model mimicking in vivo conditions, and looked for synergic combination to boost apoptosis.MethodsWe evaluated the apoptotic rate of multiple AML cell lines and AML primary cells treated with MEDS433 or other DHODH inhibitors, alone and in combination with classical antileukemic drugs or with dipyridamole, a blocker of the pyrimidine salvage pathway. Experiments were also performed mimicking in vivo conditions, i.e., in the presence of physiological uridine plasma levels (5 μM).ResultsMEDS433 showed a strong apoptotic effect against multiple AML cell lines, which was at least partially independent from the differentiation process. Its combination with classical antileukemic agents resulted in a further increase of the apoptotic rate. However, when MEDS433 was tested in the presence of 5 μM uridine and/or in primary AML cells, results were less impressive. On the contrary, the combination of MEDS433 with dipyridamole resulted in an outstanding synergistic effect, with a dramatic increase of the apoptotic rate both in AML cell lines and AML primary cells, which was unaffected by physiological uridine concentrations. Preliminary analyses show that the toxicity of this treatment should be limited to proliferating cells.ConclusionsThe combination of a DHODH inhibitor and dipyridamole is characterized by differentiating and pro-apoptotic features and induces metabolic lethality on a wide variety of AMLs with different genetic backgrounds.

Maize Thymidine Kinase Activity Is Present throughout Plant Development and Its Heterologous Expression Confers Tolerance to an Organellar DNA-Damaging Agent

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers

ABSTRACT Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum. Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering. IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus. Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase (DHODH) as its target, along with the discovery that knockdown of genes in pyrimidine salvage pathways sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was efficiently converted into its triphosphates in cells. When combined with GSK983, it led to large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral genome infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activities of RdRp inhibitors.

Multiplex genetic engineering exploiting pyrimidine salvage pathway-based self-encoded selectable markers

ABSTRACTSelectable markers are indispensable for genetic engineering, yet their number and variety is limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs-of-interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, growth or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use inserting three fluorescent protein encoding genes into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum.Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.

Contribution of Cytidine Deaminase to Thymidylate Biosynthesis in Trypanosoma brucei: Intracellular Localization and Properties of the Enzyme

ABSTRACT Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of Trypanosoma brucei CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2′-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs T. brucei proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for de novo thymidylate biosynthesis. This observation contrasts with the existence in T. brucei of a dimeric deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, T. brucei dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in T. brucei, CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism. IMPORTANCE Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5′-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.