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

Thymark, Majbritt; Johansson, E.; Larsen, Sine; Willemoës, Martin

doi:10.1016/j.abb.2007.10.013

Cited by 8 publications

(7 citation statements)

References 26 publications

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“…The dcd mutants described here have an MIC of vancomycin of 50 g/ml, suggesting that small-molecule inhibitors of DCD would have the same effect and would be useful potentiators. The three-dimensional (3D) structure of E. coli DCD is known (41,58), facilitating drug design. The E. coli DCD enzyme shares extensive homology among Gram-negative bacteria.…”

Section: Discussionmentioning

confidence: 99%

Deoxycytidine Deaminase-Deficient Escherichia coli Strains Display Acute Sensitivity to Cytidine, Adenosine, and Guanosine and Increased Sensitivity to a Range of Antibiotics, Including Vancomycin

Kang¹,

Yuan²,

Zhou³

et al. 2014

Journal of Bacteriology

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We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin.

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Section: Discussionmentioning

confidence: 99%

Deoxycytidine Deaminase-Deficient Escherichia coli Strains Display Acute Sensitivity to Cytidine, Adenosine, and Guanosine and Increased Sensitivity to a Range of Antibiotics, Including Vancomycin

Kang¹,

Yuan²,

Zhou³

et al. 2014

Journal of Bacteriology

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“…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 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). 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).…”

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confidence: 99%

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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“…In the investigated enzymes the regulation pattern showed some species specific differences however, the following characteristics were observed for all of the investigated enzymes: (i) the reaction shows kinetic cooperativity if investigated under steady-state conditions 49,50,73 , (ii) the enzyme activity is inhibited by dTTP binding to the active site, but the mechanism of inhibition is more complicated than simple competition 49,50,63,73,90 , (iii) dTTP binds to an inactive conformation of the active site, (iv) the inactive conformation of one active site forces the same conformation to the other active sites 49,50 , (v) pre-steady-state experiments show that product formation is preceded by a time lag, which was shown to be prolonged upon the addition of dTTP 49,63 . The binding of dTTP is possible only to the inactive conformation, because the additional metal group of the thymine ring cannot be accommodated in the active conformation 49,50 .…”

Section: Regulation Of the Dctp Deaminase Activitymentioning

confidence: 97%

Investigation of the traditional enzymatic role and the emerging regulatory function of dUTPases

Szabó¹

2015

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

Cited by 8 publications

References 26 publications

Deoxycytidine Deaminase-Deficient Escherichia coli Strains Display Acute Sensitivity to Cytidine, Adenosine, and Guanosine and Increased Sensitivity to a Range of Antibiotics, Including Vancomycin

Deoxycytidine Deaminase-Deficient Escherichia coli Strains Display Acute Sensitivity to Cytidine, Adenosine, and Guanosine and Increased Sensitivity to a Range of Antibiotics, Including Vancomycin

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

Investigation of the traditional enzymatic role and the emerging regulatory function of dUTPases

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