Fig 1.
a) The radical generation and transfer pathways of all three classes of RNR are thought to converge on a completely conserved cysteine residue that transfers it to the substrate. The class I, II and III enzymes are coloured mauve, pink and gold respectively. The finger loops of all three classes and the C-terminal loop of the class III RNRs, as exemplified by the enzyme from bacteriophage T4, are shown in cartoon representation. The position of the glycyl radical in class III is marked by a sphere. The two hydrogen-bonded Tyr residues that end the proton-coupled electron transfer chain (PCET) in class I are shown in mauve, with the terminal oxygen atom shown as a sphere. The 5’-deoxyadenosine moiety generated by cleavage of the C-Co bond in AdoCbl by class II RNRs is shown with the 5’-C atom shown as a pink sphere. The GDP substrate bound to the class II enzyme is shown as sticks with the C3’ atom marked with a gray sphere. b) Overall structure of the tmNrdD dimer. The left-hand monomer is coloured grey while the right-hand monomer is coloured as a spectrum from deep blue at the N-terminus to deep red at the C-terminus. The allosteric effector dATP and the substrate CTP are shown in space-filling representation. c) Comparison of the structures of tmNrdD and the previously determined structure of NrdD from bacteriophage T4 [9]. The T4 structure is coloured dark blue and tmNrdD is coloured red. d) Depiction of the active site area where the tips of the finger loop (blue) and the C-terminal loop (orange) meet. The position of the glycyl radical is marked by an orange sphere and Ile359 at the tip of the finger loop by a blue sphere. The substrate CTP is shown in stick representation. The Zn-binding domain is shown in yellow.
Fig 2.
a) Details of the conformation of the glycyl radical and finger loops in the active site of tmNrdD. The two loops are shown as sticks with the surrounding β-barrel as a cartoon. A 2m|Fo|-D|Fc| omit electron density map is shown contoured at 1.0 σ. The map was calculated by omitting residues 351–364 and 618–623 from the model followed by three macrocycles of torsion angle molecular dynamics in phenix.refine with default parameters. b) Cross-eyed stereo view comparing the glycyl radical loops in several representative glycyl radical enzymes. tmNrdD is shown in red, T4NrdD in blue, pyruvate formate lyase in magenta, glycerol dehydratase in green and 4-hydroxyphenylacetate decarboxylase in orange. The Cα atom of Gly621 in tmNrdD is indicated with a red sphere.
Fig 3.
Position of the SCCR motif in relation to the active site in tmNrdD.
The C-terminal domain and glycyl radical loop are shown in orange and the finger loop in light blue, with the Gly and Ile residues at their respective tips shown as spheres. The two residues Ser and Cys at the beginning of the SCCR motif are also pinpointed by spheres. The preceding β-strand βE is drawn in yellow and the approximate location of a disordered 20-residue segment following the SCCR motif is sketched. For clarity several α-helices on the front of the barrel have been removed.
Fig 4.
a) Details of the interactions of the allosteric effector dATP. The Mg2+ ion is shown as a green sphere. Monomer A of tmNrdD is coloured orange and monomer B is coloured grey. Hydrogen bonds to the protein are shown as dotted lines. An anomalous difference map generated by substitution of the Mg2+ ion by Mn2+ and collection of data near the Mn2+ K-edge (1.86 Å) is shown as green chicken wire. The contour level is 5.0 σ. An m|Fo|-D|Fc| omit map generated by excluding the coordinates of dATP followed by torsion angle simulated annealing in phenix.refine is shown as grey mesh, contoured at 3.0 σ. b) Communication pathway between allosteric effector dATP and substrate CTP. Both are shown in stick representation, as are the critical side chains that read out the effector base and those that make H-bonds to the substrate. Colouring is as in panel a) but loop 2 is coloured a darker shade of gray. c) Overall view of the interactions of CTP with tmNrdD. Hydrogen bonds are shown as dotted lines and water molecules involved in the network as small red spheres. The water molecule that lies between the CTP 2’-OH group and Cys125 is coloured green and the one in the bend of the triphosphate moiety discussed in the text is coloured pale blue. d) Close-up of the interactions of the CTP phosphate moieties with tmNrdD. Important hydrogen bonds are shown as dark dotted lines and water molecules involved in the interactions as small red spheres.
Fig 5.
Schematic of key distances between the Cα atom of Gly621, atoms in Ile359 and the H3’ atom of the substrate CTP.
Distances between key atoms are shown in Å.
Fig 6.
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.
Table 1.
Activity assays on mutants of cysteine residues in the SCCR motif.
Fig 7.
Unlabelled phylogenetic tree for the NrdDh class of NrdD sequences.
The characteristic sequence motifs for each of the subgroups NrdDh1-4 are shown, along with the phyla. For more details, including the presence of other RNR classes in the genomes of the organisms, see S6 Fig.