Journal: Applied and Environmental Microbiology

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

doi: 10.1128/AEM.00268-15

Figure Lengend Snippet: Aerobic deoxyribonucleotide metabolism, including the regulatory patterns (dotted lines). + and −, activation and inhibition, respectively. The alternative pathways from dCTP/dCMP to dTTP are shown using bold arrows, and both the dcdB- and comEB-dependent pathways are present in B. halodurans. Gene designations are as follows: nrdAB, aerobic ribonucleotide reductase; ndk, nucleoside diphosphate kinase; dcd, monofunctional dCTP deaminase; dut, dUTPase; dcdB, DCD:DUT; comEB, dCMP deaminase; thyA, thymidylate synthase; thyX, NADPH-dependent thymidylate synthase; tdk, thymidine kinase; cdd, cytidine deaminase; tmk, dTMP kinase.

Article Snippet: We thank Preben Nielsen (Novozymes) for the Bacillus halodurans C125 strain and MAXLab for beam time.

Techniques: Activation Assay, Inhibition

Journal: Applied and Environmental Microbiology

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

doi: 10.1128/AEM.00268-15

Figure Lengend Snippet: Primers used for qPCR quantification of gene expression in B. halodurans strain C125

Article Snippet: We thank Preben Nielsen (Novozymes) for the Bacillus halodurans C125 strain and MAXLab for beam time.

Techniques: Gene Expression, Sequencing

Journal: Applied and Environmental Microbiology

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

doi: 10.1128/AEM.00268-15

Figure Lengend Snippet: Analysis of the B. halodurans DCD:DUT reaction. Progress curves of[5-3H]dUMP formation from [5-3H]dCTP (A) and deamination of dCTP (B) are shown. Assays were performed as described in Materials and Methods in the presence 0.4 mM [5-3H]dCTP or 0.4 mM dCTP, both at pH 6.8. The enzyme concentrations were 3.4 μM (closed circles) and 1.7 μM (open circles).

Article Snippet: We thank Preben Nielsen (Novozymes) for the Bacillus halodurans C125 strain and MAXLab for beam time.

Techniques:

Journal: Applied and Environmental Microbiology

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

doi: 10.1128/AEM.00268-15

Figure Lengend Snippet: Substrate saturation and inhibition of B. halodurans dCMP deaminase and DCD:DUT by dTTP. Assays were performed as described in Materials and Methods by determining the change in absorption at 291 nm over time. (A) Saturation of dCMP deaminase with dCMP in the presence of 100 μM dCTP at pH 7.5. Data were fitted to equation 1. The kinetic parameters were as follows: S0.5 = 0.22 ± 0.03 mM, kcat = 1.9 ± 0.1 s−1, and nH = 1.27 ± 0.08. (B) Saturation of DCD:DUT with dCTP at pH 6.8. Data were fitted to equation 1. The kinetic parameters were as follows: S0.5 = 0.047 ± 0.003 mM, kcat = 0.28 ± 0.01 s−1, and nH = 2.3 ± 0.3. (C) Inhibition by dTTP at pH 7.5 of dCMP deaminase in the presence of 100 μM dCMP and 100 μM dCTP (open circles) or 20 μM dCMP and 100 μM dCTP (triangles) and DCD:DUT (closed circles) in the presence of 100 μM dCTP and 100 μM dCMP. Data were fitted to equation 2 (open circles, triangles) or equation 3 (closed circles). The kinetic parameters were as follows: rateuninh = 0.20 ± 0.1 s−1, rateoffset = 0.12 ± 0.01 s−1, I0.5 = 0.012 ± 0.001 mM, and nH = 1.8 ± 0.3 (open circles); rateuninh = 0.09 ± 0.01 s−1, rateoffset = 0.052 ± 0.009 s−1, I0.5 = 0.008 ± 0.002 mM, and nH = 2.0 ± 1.0 (triangles); and rateuninh = 0.208 ± 0.009 s−1, I0.5 = 0.030 ± 0.003 mM, and nH = 1.6 ± 0.2 (closed circles). (D) Activation by dCTP at pH 7.5 of dCMP deaminase in the presence of 100 μM dCMP (open circles) or 20 μM dCMP (triangles) and saturation of DCD:DUT with dCTP in the presence of 100 μM dCMP (closed circles). Data were fitted to equation 1. The kinetic parameters were as follows: A0.5 = 0.07 ± 0.02 mM, kcat = 0.73 ± 0.01 s−1, and nH = 1.1 ± 0.1 (open circles); A0.5 = 0.07 ± 0.04 mM, kcat = 0.18 ± 0.04 s−1, and nH = 1.0 ± 0.2 (triangles); and S0.5 = 0.025 ± 0.003 mM, kcat = 0.20 ± 0.01 s−1, and nH = 1.8 ± 0.3 (closed circles).

Article Snippet: We thank Preben Nielsen (Novozymes) for the Bacillus halodurans C125 strain and MAXLab for beam time.

Techniques: Inhibition, Activation Assay

Journal: Applied and Environmental Microbiology

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

doi: 10.1128/AEM.00268-15

Figure Lengend Snippet: (A) Overall fold of the DCD:DUT from B. halodurans. The coloring progresses from the N terminus (blue) to the C terminus (red). (B) Binding of dTTP is clearly seen in a 2Fo − Fc omit map contoured at a level of 0.8σ. (C) Schematic view of the hydrogen bonds to dTTP in DCD:DUT from B. halodurans. (D) A closeup on the nucleotide binding site, where all structures are superposed. Blue, the E. coli dCTP deaminase monomer (PDB accession number 1XS1; dUTP bound); purple, the M. jannaschii DCD:DUT monomer (PDB accession number 2HXD; E145A variant, dUMP NP bound); green, the M. tuberculosis DCD:DUT monomer (PDB accession number 2QXX; dTTP bound); yellow, the S. tokodaii putative DCD:DUT monomer (PDB accession number 2YZJ; dUDP bound); red, the B. halodurans DCD:DUT monomer (PDB accession number 4XJC; dTTP bound). Panels A, B, and D were prepared using the PyMOL molecular graphics system.

Article Snippet: We thank Preben Nielsen (Novozymes) for the Bacillus halodurans C125 strain and MAXLab for beam time.

Techniques: Binding Assay, Variant Assay

Journal: Applied and Environmental Microbiology

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

doi: 10.1128/AEM.00268-15

Figure Lengend Snippet: Comparison of crystal structures of DCD:DUT from B. halodurans and dCTP deaminases and bifunctional DCD:DUT enzymes from different organisms

Article Snippet: We thank Preben Nielsen (Novozymes) for the Bacillus halodurans C125 strain and MAXLab for beam time.

Techniques: Comparison