ABSTRACT
The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.
Subject(s)
Antigens, Bacterial/biosynthesis , Carbohydrate Epimerases , Coxiella burnetii/enzymology , Streptomyces griseus/enzymology , Carbohydrate Epimerases/genetics , Nucleoside Diphosphate Sugars/biosynthesis , Thymine Nucleotides/biosynthesisABSTRACT
Disturbed deoxythymidine triphosphate biosynthesis due to the inhibition of thymidylate synthase (TS) can lead to uracil accumulation in DNA, eventually, lead to neurocytes apoptosis and cognitive decline. Folic acid supplementation delayed cognitive decline and neurodegeneration in senescence-accelerated mouse prone 8 (SAMP8). Whether folic acid, one of nutrition factor, the effect on the expression of TS is unknown. The study aimed to determine if folic acid supplementation could alleviate age-related cognitive decline and apoptosis of neurocytes by increasing TS expression in SAMP8 mice. According to folic acid concentration in diet, four-month-old male SAMP8 mice were randomly divided into three different diet groups by baseline body weight in equal numbers. Moreover, to evaluate the role of TS, a TS inhibitor was injected intraperitoneal. Cognitive test, apoptosis rates of neurocytes, expression of TS, relative uracil level in telomere, and telomere length in brain tissue were detected. The results showed that folic acid supplementation decreased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, alleviated telomere length shorting, increased expression of TS, then decreased apoptosis rates of neurocytes, and alleviated cognitive performance in SAMP8 mice. Moreover, at the same concentration of folic acid, TS inhibitor raltitrexed increased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, and exacerbated telomere length shorting, decreased expression of TS, then increased apoptosis rates of neurocytes, and decreased cognitive performance in SAMP8 mice. In conclusion, folic acid supplementation alleviated age-related cognitive decline and inhibited apoptosis of neurocytes by increasing TS expression in SAMP8 mice.
Subject(s)
Aging , Brain/metabolism , Cognitive Dysfunction/diet therapy , Dietary Supplements , Folic Acid/administration & dosage , Neurons/physiology , Thymine Nucleotides/biosynthesis , Animals , Apoptosis , Folic Acid/blood , Folic Acid/metabolism , Male , Memory , Mice , Morris Water Maze Test , Quinazolines/pharmacology , Telomere Shortening , Thiophenes/pharmacology , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism , Uracil/metabolismABSTRACT
Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC50 of 120-410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Hydro-Lyases/metabolism , Nucleoside Diphosphate Sugars/biosynthesis , Racemases and Epimerases/metabolism , Streptococcus/enzymology , Thymine Nucleotides/biosynthesis , Anti-Bacterial Agents/pharmacology , Biosynthetic Pathways , Hydro-Lyases/genetics , Inhibitory Concentration 50 , Racemases and Epimerases/genetics , Streptococcus/drug effectsABSTRACT
Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.
Subject(s)
Folic Acid/metabolism , Mitochondria/metabolism , Protein Biosynthesis , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Aminohydrolases/metabolism , Biocatalysis , Carrier Proteins/metabolism , Codon/genetics , Electron Transport , Folic Acid Antagonists/pharmacology , GTP-Binding Proteins/metabolism , Glycine Hydroxymethyltransferase/deficiency , Glycine Hydroxymethyltransferase/metabolism , Guanosine/metabolism , HCT116 Cells , HEK293 Cells , Humans , Leucine/genetics , Lysine/genetics , Methylation/drug effects , Methylenetetrahydrofolate Dehydrogenase (NADP)/metabolism , Mitochondria/drug effects , Mitochondria/enzymology , Multifunctional Enzymes/metabolism , Oxidative Phosphorylation/drug effects , RNA, Transfer/genetics , RNA-Binding Proteins , Ribosomes/metabolism , Sarcosine/metabolism , Tetrahydrofolates/metabolism , Thymine Nucleotides/biosynthesisABSTRACT
Members of the dUTPase superfamily play an important role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. dCTP deaminases and the bifunctional dCTP deaminase-dUTPases are cooperatively regulated by dTTP. However, the manifestation of allosteric behavior within the same trimeric protein architecture of dUTPases, the third member of the superfamily, has been a question of debate for decades. Therefore, we designed hybrid dUTPase trimers to access conformational states potentially mimicking the ones observed in the cooperative relatives. We studied how the interruption of different steps of the enzyme cycle affects the active site cross talk. We found that subunits work independently in dUTPase. The experimental results combined with a comparative structural analysis of dUTPase superfamily enzymes revealed that subtile structural differences within the allosteric loop and the central channel in these enzymes give rise to their dramatically different cooperative behavior. We demonstrate that the lack of allosteric regulation in dUTPase is related to the functional adaptation to more efficient dUTP hydrolysis which is advantageous in uracil-DNA prevention.
Subject(s)
DNA/metabolism , Pyrophosphatases/metabolism , Uracil/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Circular Dichroism , Humans , Kinetics , Magnesium/chemistry , Magnesium/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleotide Deaminases/genetics , Nucleotide Deaminases/metabolism , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Secondary , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , Sequence Alignment , Spectrometry, Fluorescence , Thymine Nucleotides/biosynthesisSubject(s)
Anemia, Megaloblastic/chemically induced , Folic Acid/metabolism , Vitamin B 12/metabolism , Anemia, Megaloblastic/metabolism , DNA/biosynthesis , Gastrointestinal Absorption/drug effects , Humans , Purines/biosynthesis , Pyrimidines/biosynthesis , Thymine Nucleotides/biosynthesis , Vitamin B 12/pharmacokinetics , Vitamin B 12 Deficiency/chemically inducedABSTRACT
The sugar nucleotide dTDP-L-rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four-step dTDP-L-rhamnose biosynthesis pathway is catalyzed by dTDP-4-dehydrorhamnose reductases (RmlD). RmlD from the Gram-negative bacterium Salmonella is the only structurally characterized family member and requires metal-dependent homo-dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram-negative and Gram-positive RmlD homologues predicts that enzymes from all Gram-positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn-sequencing and generation of a conditional-expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram-positive bacteria and a subset of Gram-negative bacteria. These results will help future screens for novel inhibitors of dTDP-L-rhamnose biosynthesis.
Subject(s)
Bacterial Proteins/metabolism , Carbohydrate Dehydrogenases/genetics , Carbohydrate Dehydrogenases/metabolism , Streptococcus pyogenes/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carbohydrate Dehydrogenases/chemistry , Carbohydrate Epimerases/metabolism , Cloning, Molecular , Gram-Positive Bacteria/enzymology , High-Throughput Nucleotide Sequencing , Humans , Models, Molecular , Mutation , Nucleoside Diphosphate Sugars/biosynthesis , Protein Structure, Tertiary , Rhamnose/analogs & derivatives , Rhamnose/biosynthesis , Rhamnose/metabolism , Sequence Alignment , Streptococcus pyogenes/genetics , Thymine Nucleotides/biosynthesis , Thymine Nucleotides/metabolismABSTRACT
The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.
Subject(s)
Mitochondria, Heart/metabolism , Myocardium/metabolism , Thymidine Kinase/metabolism , Thymidine Monophosphate/biosynthesis , Thymine Nucleotides/biosynthesis , Animals , Carbonyl Cyanide m-Chlorophenyl Hydrazone/analogs & derivatives , Carbonyl Cyanide m-Chlorophenyl Hydrazone/chemistry , Creatine Kinase/chemistry , Cytosol/metabolism , Electron Transport , Female , Membrane Potential, Mitochondrial , Oligomycins/chemistry , Phosphocreatine/chemistry , Phosphorylation , Potassium Cyanide/chemistry , Rats , Rats, Sprague-Dawley , Thymidine/metabolism , Zidovudine/pharmacologyABSTRACT
In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.
Subject(s)
Cell Nucleus/genetics , DNA Repair , Mitochondria/enzymology , Stress, Physiological/genetics , Thymidine Kinase/physiology , Thymine Nucleotides/biosynthesis , Cell Cycle , Cell Line , DNA Breaks, Double-Stranded , DNA Damage , Deoxyribonucleotides/metabolism , Genome , Humans , Intracellular Signaling Peptides and Proteins/analysis , Thymidine Kinase/metabolism , Tumor Suppressor p53-Binding Protein 1 , Ultraviolet RaysABSTRACT
Synthetic methods were investigated for the preparation of O and S-glucosyl thiophosphates and glucosyl 1C-thiophosphonate. Four protected glucosyl thiophosphate compounds were synthesized and characterized as precursors to glucose 1-thiophosphate. The effect of various reaction conditions and the nature of the carbohydrate and thiophosphate protecting groups and how they impact both the yields and α/ß diastereoselectivity of the glucosyl thiophosphate products were explored. A novel isomerization from an O-linked to S-linked glucosyl thiophosphate was observed. α-D-Glucose-1C-thiophosphonate was synthesized and evaluated as a substrate for the thymidylyltransferase, Cps2L. Tandem mass spectrometric analysis determined the position of sulfur in the sugar nucleotide product.
Subject(s)
Glucose/analogs & derivatives , Glucosephosphates/chemistry , Glucosephosphates/metabolism , Nucleotidyltransferases/metabolism , Phosphates/metabolism , Thymine Nucleotides/biosynthesis , Thymine Nucleotides/metabolism , Carbohydrate Conformation , Enzyme Activation , Glucose/biosynthesis , Glucose/chemistry , Glucose/metabolism , Glucosephosphates/chemical synthesis , Phosphates/chemistry , Tandem Mass Spectrometry , Thymine Nucleotides/chemistryABSTRACT
Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-D-allose. Four enzymes are required for the production of dTDP-6-deoxy-D-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-D-allose formation. Specifically, it has been proposed that ChmJ is a 3'-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using (1)H nuclear magnetic resonance that ChmJ, indeed, functions as a 3'-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono- or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft.
Subject(s)
Nucleoside Diphosphate Sugars/biosynthesis , Racemases and Epimerases/metabolism , Thymine Nucleotides/biosynthesis , Base Sequence , Crystallography, X-Ray , DNA Primers , Kinetics , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Racemases and Epimerases/chemistryABSTRACT
Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP â¼30-fold and decreases dTTP â¼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.
Subject(s)
DCMP Deaminase/genetics , DNA Replication/genetics , Genomic Instability , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Cell Cycle/genetics , Checkpoint Kinase 1 , DCMP Deaminase/deficiency , DNA Damage , DNA Helicases/metabolism , DNA Repair/genetics , Deoxycytosine Nucleotides/biosynthesis , Nucleotidyltransferases/metabolism , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Thymine Nucleotides/biosynthesisABSTRACT
Pseudomonas aeruginosa produces as biosurfactants rhamnolipids, containing one (mono-rhamnolipid) or two (di-rhamnolipid) l-rhamnose molecules. The rhamnosyltransferase RhlB catalyses the synthesis of mono-rhamnolipid using as precursors dTDP-l-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) produced by RhlA, while the rhamnosyltransferase RhlC synthesizes di-rhamnolipid using mono-rhamnolipid and dTDP-l-rhamnose as substrates. The Las and Rhl quorum-sensing systems coordinately regulate the production of these surfactants, as well as that of other exoproducts involved in bacterial virulence, at the transcriptional level in a cell density-dependent manner. In this work we study the transcriptional regulation of the rmlBDAC operon, encoding the enzymes involved in the production of dTDP-l-rhamnose, the substrate of both rhamnosyltransferases, RhlB and RhlC, and also a component of P. aeruginosa lipopolysaccharide. Here we show that the rmlBDAC operon possesses three promoters. One of these transcriptional start sites (P2) is responsible for most of its expression and is dependent on the stationary phase sigma factor σ(S) and on RhlR/C(4)-HSL through its binding to an atypical 'las box'.
Subject(s)
Bacterial Proteins/metabolism , Operon , Pseudomonas aeruginosa/genetics , Quorum Sensing/genetics , Sigma Factor/metabolism , Bacterial Proteins/genetics , Base Sequence , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Nucleoside Diphosphate Sugars/biosynthesis , Promoter Regions, Genetic , Pseudomonas aeruginosa/enzymology , Sigma Factor/genetics , Thymine Nucleotides/biosynthesis , Transcription Initiation SiteABSTRACT
Pasteurella multocida strains are classified into 16 different lipopolysaccharide (LPS) serovars using the Heddleston serotyping scheme. Ongoing studies in our laboratories on the LPS aim to determine the core oligosaccharide (OS) structures expressed by each of the Heddleston type strains and identify the genes and transferases required for the biosynthesis of the serovar-specific OSs. In this study, we have determined the core OS of the LPS expressed by the Heddleston serovar 9 type strain, P2095. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy and mass spectrometry revealing the following structure: . The serovar 9 OS contains an inner core that is conserved among P. multocida strains with an elaborate outer core extension containing rhamnose (Rha), a D-glycero-D-manno isomer of heptose, and the unusual deoxyamino sugar, 3-acetamido-3,6-dideoxy-α-D-glucose (Qui3NAc). Genetic analyses of the LPS outer core biosynthesis locus revealed that in addition to the glycosyltransferases predicted to transfer the sugars to the nascent LPS molecule, the locus also contained the complete set of genes required for the biosynthesis of the nucleotide sugar donors dTDP-Rha and dTDP-Qui3NAc. One of the genes identified as part of the dTDP-Qui3NAc biosynthesis pathway, qdtD, encodes a proposed bi-functional enzyme with N-terminal amino acid identity to dTDP-4-oxo-6-deoxy-D-glucose-3,4-oxoisomerase and C-terminal amino acid identity to dTDP-3-oxo-6-deoxy-α-D-glucose transacetylase.
Subject(s)
Bacterial Proteins/genetics , Deoxy Sugars/biosynthesis , Lipopolysaccharides/chemistry , Pasteurella multocida/enzymology , Thymine Nucleotides/biosynthesis , Amino Acid Sequence , Carbohydrate Conformation , Carbohydrate Sequence , Genes, Bacterial , Genetic Loci , Glycosyltransferases/genetics , Mass Spectrometry , Molecular Sequence Data , Pasteurella multocida/genetics , Sequence Homology, Amino AcidABSTRACT
The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.
Subject(s)
Biosynthetic Pathways , Mammals/metabolism , Mitochondria/metabolism , Thymine Nucleotides/biosynthesis , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , DNA, Mitochondrial/metabolism , Gene Expression Regulation , Glycine/metabolism , Humans , Mitochondria/enzymology , Molecular Sequence Data , Protein Transport , Sequence Alignment , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism , Thymidine Monophosphate/biosynthesis , Thymidylate Synthase/metabolism , Uracil/metabolismABSTRACT
Folate-mediated one-carbon metabolism is required for the de novo synthesis of purines, thymidylate, and S-adenosylmethionine, the primary cellular methyl donor. Impairments in folate metabolism diminish cellular methylation potential and genome stability, which are risk factors for colorectal cancer (CRC). Cytoplasmic serine hydroxymethyltransferase (SHMT1) regulates the partitioning of folate-activated one-carbons between thymidylate and S-adenosylmethionine biosynthesis. Therefore, changes in SHMT1 expression enable the determination of the specific contributions made by thymidylate and S-adenosylmethionine biosynthesis to CRC risk. Shmt1 hemizygosity was associated with a decreased capacity for thymidylate synthesis due to downregulation of enzymes in its biosynthetic pathway, namely thymidylate synthase and cytoplasmic thymidine kinase. Significant Shmt1-dependent changes to methylation capacity, gene expression, and purine synthesis were not observed. Shmt1 hemizygosity was associated with increased risk for intestinal cancer in Apc(min)(/+) mice through a gene-by-diet interaction, indicating that the capacity for thymidylate synthesis modifies susceptibility to intestinal cancer in Apc(min)(/+) mice.
Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Glycine Hydroxymethyltransferase/genetics , Intestinal Neoplasms/genetics , Intestinal Neoplasms/metabolism , Thymine Nucleotides/biosynthesis , Adenomatous Polyposis Coli Protein/metabolism , Animals , Blotting, Western , Cells, Cultured , Colon/metabolism , Colon/pathology , Diet , Enterocytes/metabolism , Epithelial Cells/metabolism , Female , Folic Acid/metabolism , Gene Expression Profiling , Glycine Hydroxymethyltransferase/metabolism , Heterozygote , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Oligonucleotide Array Sequence Analysis , Purines/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Risk FactorsABSTRACT
Uracil may occur in DNA due to either cytosine deamination or thymine replacing incorporation. Its quantitative characterization is important in assessing DNA damages in cells with perturbed thymidylate metabolism or within different DNA segments involved in immunoglobulin gene diversification. The archaeal DNA polymerase from Pyrococcus furiosus binds strongly to the deaminated base uracil and stalls on uracil-containing templates. Here, we present a straightforward method for quantitative assessment of uracil in DNA within specific genomic segments. We use wild-type P. furiosus polymerase in parallel with its point mutant version which lacks the uracil-binding specificity on synthetic and genomic DNA samples to quantify the uracil content in a single-step real-time PCR assay. Quantification of the PCR results is based on an approach analogous to template copy number determination in comparing different samples. Data obtained on synthetic uracil-containing templates are verified by direct isotopic measurements. The method is also tested on physiological DNA samples from Escherichia coli and mouse cell lines with perturbed thymidylate biosynthesis. The present PCR-based method is easy to use and measures the uracil content within a genomic segment defined by the primers. Using distinct sets of primers, the method allows the analysis of heterogeneity of uracil distribution within the genome.
Subject(s)
DNA/chemistry , Polymerase Chain Reaction/methods , Uracil/analysis , Animals , Cell Line , DNA, Bacterial/chemistry , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Deoxyuridine/analysis , Escherichia coli/genetics , Genome, Bacterial , Mice , Plasmids/genetics , Point Mutation , Pyrococcus furiosus/enzymology , Thymine Nucleotides/biosynthesisABSTRACT
Biosynthetic genes encoding proteins involved in the first steps of deoxyhexose biosynthesis from D-glucose-1-phosphate were expressed in Saccharopolyspora erythraea. The resulting mutant was able to accumulate and utilise TDP-L-olivose. Co-expression of the spinosyn glycosyl transferase SpnP in the resulting mutant endowed upon it the ability to biotransform exogenously added spinosyn aglycones to yield novel spinosyn analogues.
Subject(s)
Deoxy Sugars/biosynthesis , Insecticides/chemical synthesis , Insecticides/pharmacology , Macrolides/chemical synthesis , Saccharopolyspora/genetics , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Deoxy Sugars/pharmacology , Gene Expression Regulation, Bacterial , Glycosylation , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Insecta/drug effects , Insecta/physiology , Insecticides/chemistry , Lethal Dose 50 , Macrolides/pharmacology , Saccharopolyspora/enzymology , Thymine Nucleotides/biosynthesisABSTRACT
3-Acetamido-3,6-dideoxy-alpha-d-glucose or Quip3NAc is an unusual deoxyamino sugar found in the O-antigens of some Gram-negative bacteria and in the S-layers of Gram-positive bacteria. It is synthesized in these organisms as a dTDP-linked sugar via the action of five enzymes. The focus of this investigation is on QdtB from Thermoanaerobacterium thermosaccharolyticum E207-71, a PLP-dependent aminotransferase that catalyzes the penultimate step in the production of dTDP-Quip3NAc. For this analysis, the enzyme was crystallized in the presence of its product, dTDP-Quip3N, and the structure was solved and refined to 2.15 A resolution. QdtB is a dimer, and its overall fold places it into the well-characterized aspartate aminotransferase superfamily. Electron density corresponding to the bound product reveals the presence of a Schiff base between C-4' of the PLP cofactor and the amino nitrogen of the sugar. Those amino acid side chains involved in binding the dTDP-sugar into the active site include Tyr 183, His 309, and Tyr 310 from subunit 1 and Lys 219 from subunit 2. Notably there is a decided lack of interactions between the pyranosyl C-4' hydroxyl of the dTDP-sugar and the protein. In keeping with this observation, we show that QdtB can also turn over dTDP-3-acetamido-3,6-dideoxy-alpha-d-galactose. This investigation represents the first structural analysis of a sugar-modifying aminotransferase with a bound product in its active site that functions at the C-3' rather than the C-4' position of the hexose.
Subject(s)
Deoxy Sugars/biosynthesis , Thymine Nucleotides/biosynthesis , Transaminases/chemistry , Crystallization , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Protein Conformation , Schiff Bases , Spectrometry, Mass, Electrospray Ionization , Thermoanaerobacterium/enzymology , Transaminases/metabolismABSTRACT
Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with d-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structure-activity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency.