Fig 1.
An overview of the ribonucleotide reductase reaction, classes, and allosteric regulation.
(A) Ribonucleotide reductase catalyzes the conversion of ribonucleotide di- or triphosphates to their corresponding deoxyribonucleotide via a catalytic cysteine radical. (B) Examples of class I, class II, and class III enzymes with their corresponding metallocofactors and radical harbors. In each structure, one specificity effector site is circled in green and one substrate site is circled in magenta. The E. coli class Ia RNR (left) is shown in the active form with the catalytic subunits (NrdA) shown in blue and the radical-generating subunits (NrdB) shown in orange/red (PDB 6W4X) [52]. Bound nucleotides (GDP substrate and TTP specificity effector) are shown as spheres. The activity effector site, which does not have any bound nucleotide in this structure, is circled in orange. The diiron-oxo center and tyrosyl radical shown below the structure are found in the radical-generating subunit and are specific to class Ia RNRs; see Table 1 for the radical harbors of other subclasses of class I RNRs. The Thermotoga maritima class II RNR (NrdJ; middle) is shown with the bound nucleotides (GDP substrate and TTP specificity effector) shown as spheres (PDB 3O0O) [34]. The S-adenosylcobalamin (B12) cofactor found in each subunit is shown as sticks and is highlighted below the structure. The Escherichia coli T4 bacteriophage class III RNR (NrdD; right) is shown with bound nucleotides (dGTP) shown as spheres (PDB 1HK8) [85]. The activase NrdG is structurally uncharacterized and contains the 4Fe-4S cluster shown below the structure. NrdG in complex with S-adenosylmethionine generates a glycyl radical is generated on the catalytic subunit via a transient interaction. (C) Allosteric regulation of RNRs that use NDPs (left) or NTPs (right) as substrates. Following reduction of nucleotides, the deoxyribonucleotide products are either first converted to dNTPs (left) or directly used (right) as specificity effectors for determining which nucleotides bind the active site for future reduction reactions (blue arrows). In some but not all RNRs, ATP acts as an overall positive activity effector and dATP at high concentrations acts as an overall negative activity effector; ATP can also act as a specificity effector in vitro and possibility in vivo. Figure modified from [78].
Table 1.
Comparison of class I, class II, and class III RNRs highlighting the differences that make a universal activity assay challenging.
Subclasses of class III RNRs are listed as NrdD1, NrdD2, and NrdD3.
Fig 2.
Overall scheme of LC-MS/MS RNR activity assay.
Either aerobically or anaerobically, a mastermix is created and a zero point is taken prior to initiation with beta (aerobic assays) or nucleotides (anaerobic assays). The reaction is allowed to proceed for 120 s with time points taken every 30 s. Inactivation of the reaction occurs by heating the sample to 95°C in a thermocycler or heat block. After inactivation, anaerobic assays are removed from the box and all assays have 1 μL of calf intestinal phosphatase (CIP) added to dephosphorylate the nucleotides at 37°C for 2 hours. The samples are then filtered through a 0.2 μm filter and transferred to mass spectrometry vials for analysis via LC-MS/MS.
Fig 3.
Measurement of RNR activity for a single nucleotide under a single condition.
(A) Generation of a dC standard curve. A multiple reaction monitoring (MRM) curve for dC (m/z 228.2 > 112.1) is extracted from the initial total ion chromatogram (TIC) and the peaks are integrated (all in triplicate; one set of standards is shown above for the TIC and MRM) to generate a standard curve that relates the area under the dC MRM peak to the nmoles dC injected. The peak in the TIC at approximately 1.4 mins corresponds to the desired dephosphorylated product (dC) whereas the peak at approximately 3.5 mins corresponds to the dephosphorylated effector (A) in this experiment; note the MRM peak for dC is at the same retention time as the dC peak in the TIC. (B) Calculation of sample activity. Another set of MRM curves are extracted and integrated from TICs for each of the time points of the assay. The integrated MRM values are then converted to nmol dC / mg NrdA by using the standard curve to calculate nmol dC injected and then dividing by the mg NrdA in each injection. The slope of the line (in nmol/mg-min; after the aforementioned has been completed in triplicate) is the activity of the protein. All reactions are done in triplicate and are shown as mean ± standard error of the mean. The above condition contained NrdA (0.1 μM dimer), NrdB (0.5 μM dimer), ATP (3000 μM), CDP (1000 μM), Trx (30 μM), TrxR (0.5 μM), and NADPH (200 μM).
Fig 4.
Activity of E. coli class Ia ribonucleotide reductase.
Each bar represents average activity +/- standard error of the mean for three replicates. NrdA ATP and dATP conditions had 0.1 μM NrdA and 0.5 μM NrdB and 3000 μM ATP or 0.175 μM dATP, respectively, whereas the NrdB condition had 3000 μM ATP, 0.1 μM NrdB, and 0.5 μM NrdA. All conditions contained CDP (1000 μM), Trx (30 μM), TrxR (0.5 μM), and NADPH (200 μM). All protein concentrations are expressed as concentration of the dimer.
Fig 5.
Activity of StNrdD class III ribonucleotide reductase enzyme.
(A) Activity assay data for ATP, dATP, and no allosteric activity effector in the presence of GTP (substrate) and TTP (specificity effector). Error bars are shown for all replicates (3 replicates per data point) but are too small to visualize for some conditions. (B) Activities calculated from assay data in (A). Each bar represents average activity +/- standard error of the mean for three replicates. All conditions contained 0.10 μM StNrdD with 1.5 ± 0.10 glycyl radicals/dimer, 1 mM substrate GTP, 1 mM specificity effector TTP, 5 mM DTT, and 12.5 mM sodium formate; the ATP condition contained 3 mM allosteric activator ATP whereas the dATP condition contained 3 mM allosteric inhibitor dATP.
Fig 6.
The use of internal standards eliminates the need for same-day standard curves.
(A) Standard curves of the same amount of dC (0–1 nmole) run on the mass spectrometer on two different days (day 1 and day 2). (B) Standard curves run on day 1 and day 2 after internal standard correction (the area under the MRM curve of the heavy [13C/15N] dC nucleoside). (C) Activity assay data for EcNrdA and EcNrdB with nmoles dC calculated after correction of both standard and sample dC integration using a heavy dC internal standard. Both assays were run on day 2 along with the day 2 standard curve used for day 2 activity calculation; day 1 standard curve used for day 1 activity calculation was run on a previous day. (D) Calculation of activity from curves in (C) with EcNrdA or EcNrdB as the limiting reagent. NrdA activity was determined to be 2000 ± 300 nmoles dC/mg-min using the day 2 standard curve to calculate activity and 2200 ± 400 nmoles dC/mg-min using the day 1 standard curve to calculate activity. NrdB activity was determined to be 8000 ± 600 nmoles dC/mg-min using the day 2 standard curve to calculate activity and 9000 ± 700 nmoles dC/mg-min using the day 1 standard curve to calculate activity. The above conditions contained NrdA (0.1 μM dimer for NrdA condition and 0.5 μM dimer for NrdB condition), NrdB (0.5 μM dimer for NrdA condition and 0.1 μM dimer for NrdB condition), ATP (3000 μM), CDP (1000 μM), Trx (30 μM), TrxR (0.5 μM), and NADPH (200 μM).
Fig 7.
Activity of E. coli class Ia ribonucleotide reductase reducing all four ribonucleotides simultaneously in the presence of specificity effectors dATP, dGTP, and TTP and activity effector ATP.
(A) Standard curves of all four ribonucleotides mixed together in concentrations of 0–100 μM (10 μL injections). (B) Activity graphs of all four ribonucleotide products being produced simultaneously over two minutes. (C) Bar graph showing production of each of the four deoxyribonucleotide products. The dU activity above condition contained Trx (30 μM), TrxR (0.5 μM), NADPH (200 μM), ATP (3000 μM), dATP (500 μM), TTP (250 μM), dGTP (100 μM), CDP (70 μM), UDP (50 μM), GDP (200 μM), ADP (110 μM), NrdA (0.1 μM dimer), and NrdB (0.5 μM dimer). Total ion chromatogram (TIC) and individual multiple reaction monitoring (MRM) curves are shown in S4 Fig.
Fig 8.
Assay data with class Ia E. coli ribonucleotide reductase measuring the reduction of all four ribonucleotide substrates simultaneously.
(A) Activity of EcNrdA in the presence of substrates, effectors, and varying amounts of dATP. (B) Activity of EcNrdA in the presence of substrates, effectors, and varying amounts of dGTP. (C) Activity of EcNrdA in the presence of substrates, effectors, and varying amounts of TTP. Each bar is representative of the average activity +/- standard error of the mean for three replicates. The values of each bar are shown in Table 2. Except for the indicated concentrations of allosteric effectors on the X-axis, components and concentrations were as follows: Trx (30 μM), TrxR (0.5 μM), NADPH (200 μM), CDP (70 μM), UDP (50 μM), GDP (200 μM), ADP (110 μM), NrdA (0.1 μM dimer), NrdB (0.5 μM dimer).
Table 2.
Average activities +/- standard error of the mean of assays graphically represented in Fig 8.
In addition to the indicated concentration of the allosteric effectors shown below in column 1, components and concentrations were as follows: Trx (30 μM), TrxR (0.5 μM), NADPH (200 μM), CDP (70 μM), UDP (50 μM), GDP (200 μM), ADP (110 μM), NrdA (0.1 μM dimer), NrdB (0.5 μM dimer).
Table 3.
Comparison of the radioactivity and coupled RNR activity assays and the LC-MS/MS assay described in this paper.