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The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site.

Aurelius O, Johansson R, Bågenholm V, Lundin D, Tholander F, Balhuizen A, Beck T, Sahlin M, Sjöberg BM, Mulliez E, Logan DT - PLoS ONE (2015)

Bottom Line: Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms.Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date.Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry & Structural Biology, Lund University, Box 124, S-221 00 Lund, Sweden.

ABSTRACT
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

No MeSH data available.


Related in: MedlinePlus

Activity assays for the T. maritima anaerobic RNR carried out in the presence of anaerobically prepared cell extracts.a) White bars show activity in the presence of fixed amounts of cell extract and MBP-tagged tmNrdG and increasing amounts of tmNrdD. Grey bars show the corresponding activity in the absence of tmNrdG. b) activity in the presence of fixed amounts of tmNrdG and tmNrdD (0.4 nmol) and increasing amounts of T. maritima cell extract. White bars show the activity in the presence of tmNrdD and grey bars the corresponding activity in the absence of tmNrdD. The results are from 3–5 experiments, some performed with different protein preparations. All values are given as the mean +/- standard deviation. Statistical analysis was performed using the standard Student T-test.
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pone.0128199.g006: Activity assays for the T. maritima anaerobic RNR carried out in the presence of anaerobically prepared cell extracts.a) White bars show activity in the presence of fixed amounts of cell extract and MBP-tagged tmNrdG and increasing amounts of tmNrdD. Grey bars show the corresponding activity in the absence of tmNrdG. b) activity in the presence of fixed amounts of tmNrdG and tmNrdD (0.4 nmol) and increasing amounts of T. maritima cell extract. White bars show the activity in the presence of tmNrdD and grey bars the corresponding activity in the absence of tmNrdD. The results are from 3–5 experiments, some performed with different protein preparations. All values are given as the mean +/- standard deviation. Statistical analysis was performed using the standard Student T-test.

Mentions: With the ribose positioned as described, Ile359 at the tip of the finger loop is positioned in between the radical-bearing residue Gly621 and the substrate (Fig 5); however it does not reveal an obvious pathway for transfer of the radical (see Discussion). The absence of Cys at a critical position in the radical transfer pathway raised the question whether tmNrdD was a functional RNR. Initially, activity assays were hampered by the fact that the native activase tmNrdG displayed very limited solubility at pH 7.0, which is required for reconstitution of its [4Fe-4S] center. In order to overcome this we generated tmNrdG tagged with maltose binding protein (MBP) and using this system in combination with photoactivation using deazaflavin we were able to generate a glycyl radical on tmNrdD (S5 Fig). This radical has a g-value of 2.0056, which is comparable to that of Gly radicals in other anaerobic RNRs from bacteriophage T4 (2.0039) [8], E. coli (2.0033) [7,39], Lactococcus lactis (2.0033) [40] and N. bacilliformis (2.0076) [23]. The glycyl radical content, estimated using a Cu2+ standard, is about 0.15 Gly° per monomer. We do not observe the fine structure seen in the EPR spectrum of the radical from N. bacilliformis [23], similar to that seen in PFL [41] and attributed to hyperfine coupling to the α-protons of adjacent residues. Presumably the difference between T. maritima and N. bacilliformis is due to differences in the local conformation of the glycyl radical loop. Generation of the glycyl radical in D2O resulted in no change in the shape of the spectrum, consistent with the fact that the hydrogen atoms of the radical Gly in NrdD are not expected to exchange with solvent [39]. Subsequent to generation of the glycyl radical we carried out anaerobic activity assays in order to probe whether the Gly radical could be transferred to the substrate. Initial trials using formate as reductant and 3H-labeled CTP as substrate were unsuccessful. However activity was obtained using anaerobically prepared extracts of T. maritima (Fig 6). These assays showed increasing triphosphate reductase activity as a function of the concentration of tmNrdD or cell extracts in the presence of fixed amounts of the other components. Control experiments omitting either tmNrdD or tmNrdG from the reaction mixture showed only negligible activity (Fig 6). Both assays tail off at the highest concentrations of the titrated component, most likely because of exhaustion of one of the non-protein assay components. The turnover number can be calculated from the data in Fig 6 and is around 0.4 s-1. Thus tmNrdD is about 5–10 times less active than the E. coli [11], Lactococcus lactis [10] and bacteriophage T4 [8] enzymes and twice as active as that of N. bacilliformis [23]. However it should be borne in mind that the assays have been done with T. maritima cell extracts and not with purified components, and that they have been done at 37°C, while the optimal growth temperature of T. maritima is 80°C [42].


The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site.

Aurelius O, Johansson R, Bågenholm V, Lundin D, Tholander F, Balhuizen A, Beck T, Sahlin M, Sjöberg BM, Mulliez E, Logan DT - PLoS ONE (2015)

Activity assays for the T. maritima anaerobic RNR carried out in the presence of anaerobically prepared cell extracts.a) White bars show activity in the presence of fixed amounts of cell extract and MBP-tagged tmNrdG and increasing amounts of tmNrdD. Grey bars show the corresponding activity in the absence of tmNrdG. b) activity in the presence of fixed amounts of tmNrdG and tmNrdD (0.4 nmol) and increasing amounts of T. maritima cell extract. White bars show the activity in the presence of tmNrdD and grey bars the corresponding activity in the absence of tmNrdD. The results are from 3–5 experiments, some performed with different protein preparations. All values are given as the mean +/- standard deviation. Statistical analysis was performed using the standard Student T-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493059&req=5

pone.0128199.g006: Activity assays for the T. maritima anaerobic RNR carried out in the presence of anaerobically prepared cell extracts.a) White bars show activity in the presence of fixed amounts of cell extract and MBP-tagged tmNrdG and increasing amounts of tmNrdD. Grey bars show the corresponding activity in the absence of tmNrdG. b) activity in the presence of fixed amounts of tmNrdG and tmNrdD (0.4 nmol) and increasing amounts of T. maritima cell extract. White bars show the activity in the presence of tmNrdD and grey bars the corresponding activity in the absence of tmNrdD. The results are from 3–5 experiments, some performed with different protein preparations. All values are given as the mean +/- standard deviation. Statistical analysis was performed using the standard Student T-test.
Mentions: With the ribose positioned as described, Ile359 at the tip of the finger loop is positioned in between the radical-bearing residue Gly621 and the substrate (Fig 5); however it does not reveal an obvious pathway for transfer of the radical (see Discussion). The absence of Cys at a critical position in the radical transfer pathway raised the question whether tmNrdD was a functional RNR. Initially, activity assays were hampered by the fact that the native activase tmNrdG displayed very limited solubility at pH 7.0, which is required for reconstitution of its [4Fe-4S] center. In order to overcome this we generated tmNrdG tagged with maltose binding protein (MBP) and using this system in combination with photoactivation using deazaflavin we were able to generate a glycyl radical on tmNrdD (S5 Fig). This radical has a g-value of 2.0056, which is comparable to that of Gly radicals in other anaerobic RNRs from bacteriophage T4 (2.0039) [8], E. coli (2.0033) [7,39], Lactococcus lactis (2.0033) [40] and N. bacilliformis (2.0076) [23]. The glycyl radical content, estimated using a Cu2+ standard, is about 0.15 Gly° per monomer. We do not observe the fine structure seen in the EPR spectrum of the radical from N. bacilliformis [23], similar to that seen in PFL [41] and attributed to hyperfine coupling to the α-protons of adjacent residues. Presumably the difference between T. maritima and N. bacilliformis is due to differences in the local conformation of the glycyl radical loop. Generation of the glycyl radical in D2O resulted in no change in the shape of the spectrum, consistent with the fact that the hydrogen atoms of the radical Gly in NrdD are not expected to exchange with solvent [39]. Subsequent to generation of the glycyl radical we carried out anaerobic activity assays in order to probe whether the Gly radical could be transferred to the substrate. Initial trials using formate as reductant and 3H-labeled CTP as substrate were unsuccessful. However activity was obtained using anaerobically prepared extracts of T. maritima (Fig 6). These assays showed increasing triphosphate reductase activity as a function of the concentration of tmNrdD or cell extracts in the presence of fixed amounts of the other components. Control experiments omitting either tmNrdD or tmNrdG from the reaction mixture showed only negligible activity (Fig 6). Both assays tail off at the highest concentrations of the titrated component, most likely because of exhaustion of one of the non-protein assay components. The turnover number can be calculated from the data in Fig 6 and is around 0.4 s-1. Thus tmNrdD is about 5–10 times less active than the E. coli [11], Lactococcus lactis [10] and bacteriophage T4 [8] enzymes and twice as active as that of N. bacilliformis [23]. However it should be borne in mind that the assays have been done with T. maritima cell extracts and not with purified components, and that they have been done at 37°C, while the optimal growth temperature of T. maritima is 80°C [42].

Bottom Line: Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms.Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date.Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry & Structural Biology, Lund University, Box 124, S-221 00 Lund, Sweden.

ABSTRACT
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

No MeSH data available.


Related in: MedlinePlus