Regulation of dCTP deaminase from <i>Escherichia coli</i> by nonallosteric dTTP binding to an inactive form of the enzyme

Johansson, E.; Thymark, Majbritt; Bynck, J.H.; Fano, Mathias; Larsen, Sine; Willemoës, Martin

doi:10.1111/j.1742-4658.2007.05945.x

Cited by 15 publications

(35 citation statements)

References 37 publications

(37 reference statements)

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“…Likewise, DCD:DUT was inhibited by dTTP (Fig. 3C), as was previously found for both the monofunctional and the bifunctional deaminase enzymes in this family (17,43). The results in Fig.…”

Section: Resultssupporting

confidence: 87%

“…The structure of the B. halodurans DCD:DUT is very similar to that of other known structures of this enzyme, and the mechanism for inhibition by dTTP seems to closely resemble that of the pre- viously characterized enzymes (17,43). The dcdB gene is distributed widely among Bacillus species, some of which are found in the Bacillus sensu lato group (B. cereus and B. thuringiensis [66]), but is apparently absent in B. subtilis and other species within the Bacillus sensu stricto group (67).…”

Section: Discussionsupporting

confidence: 56%

“…Enteric bacteria synthesize an enzyme with dCTP deaminase activity that converts dCTP into dUTP (EC 3.5.4.13), which accounts for 70 to 80% of the dUMP production (15). The dCTP deaminase is a homotrimeric enzyme structurally related to the trimeric dUTPase and part of the dCTP deaminase/dUTPase superfamily (16)(17)(18). The genomes of many Gram-positive bacteria and all eukaryotes encode a dCMP deaminase (EC 3.5.4.12) that shares no resemblance to the enteric dCTP deaminase (14,(19)(20)(21)(22)(23).…”

mentioning

confidence: 99%

“…The dCTP deaminase catalyzes dCTP deamination solely by way of amino acid residues present in the active site (16,18), while the dCMP deaminase, a hexameric enzyme which is related to other cytosine deaminating enzymes, such as cytosine deaminase (24) and cytidine deaminase (25)(26)(27)(28)(29), binds a zinc ion that directly participates in the catalytic mechanism (14,19,20,30). One feature shared by the two enzymes, dCTP and dCMP deaminase, is that they are both feedback inhibited by dTTP (17,19,(31)(32)(33)(34)(35)(36)(37)(38). In addition, the dCMP deaminase is allosterically activated by dCTP (19,33,36), the substrate of the dCTP deaminase.…”

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confidence: 99%

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Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Oehlenschlæger

Løvgreen

Reinauer

et al. 2015

Appl Environ Microbiol

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Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. The biosynthetic route to the nucleotide dUMP, the precursor of dTTP, as shown in Fig. 1, differs between organisms and sets it apart from the rest of nucleotide metabolism (1). This is in agreement with the general belief that uracil was the original DNA base-pairing partner for adenine and that the biosynthesis of dTTP from dUMP arose later in evolution (2). The deoxyribonucleotides dGTP, dATP, and dCTP are all produced in a pathway that goes through the multisubstrate enzyme ribonucleotide reductase. This enzyme is highly regulated in order to maintain the balance of deoxyribonucleotide pools (3-5). dUTP is also formed by ribonucleotide reductase, but because of the toxicity of this nucleotide (6, 7), all organisms express an enzyme with dUTPase activity that hydrolyzes dUTP to dUMP (8).The direct formation of dUMP via ribonucleotide reductase (Fig. 1) is not an efficient process, as the ribonucleotide reductase has a reduced affinity for UDP compared to the affinities of three other canonical ribonucleotides (9-12). Instead, most of the dUMP arises from deamination of cytosine deoxyribonucleotides (13-15). Enteric bacteria synthesize an enzyme with dCTP deaminase activity that converts dCTP into dUTP (EC 3.5.4.13), which accounts for 70 to 80% of the dUMP production (15). The dCTP deaminase is a homotrimeric enzyme structurally related to the trimeric dUTPase and part of the dCTP deaminase/dUTPase superfamily (16-18). The genomes of many Gram-positive bacteria and all eukaryotes encode a dCMP deaminase (EC 3.5.4.12) that shares no resemblance to the enteric dCTP de...

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“…Likewise, DCD:DUT was inhibited by dTTP (Fig. 3C), as was previously found for both the monofunctional and the bifunctional deaminase enzymes in this family (17,43). The results in Fig.…”

Section: Resultssupporting

confidence: 87%

Section: Discussionsupporting

confidence: 56%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Oehlenschlæger

Løvgreen

Reinauer

et al. 2015

Appl Environ Microbiol

Self Cite

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“…The major pathway involves phosphorylation of dCDP to dCTP, deamination of dCTP to dUTP, and hydrolysis of dUTP to dUMP. Only 20 to 30% of the cellular dUMP is supplied by hydrolysis of dUTP (29,37). The deamination of dCTP (dcd) is located at a branch point in the pyrimidine metabolic pathway.…”

mentioning

confidence: 99%

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Lee¹,

Kim²,

Kim³

et al. 2009

Appl Environ Microbiol

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Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter ؊1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA Nglycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ϳ1.2-fold (740.3 mg liter ؊1 ). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.

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Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

Thymark

Johansson

Larsen

et al. 2008

Archives of Biochemistry and Biophysics

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Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme

Cited by 15 publications

References 37 publications

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

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