Limits...
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

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493059&req=5

pone.0128199.g004: 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.

Mentions: The structure of tmNrdD has also been determined in complex with the allosteric substrate specificity effector dATP and its cognate substrate CTP (Fig 4), as well as with dATP alone. The effector dATP binds at the dimer interface, along the length of helices αA and αB of the α-β barrel (Figs 1B, 4A & 4B; the naming of secondary structure elements follows the convention for T4NrdD [9]). The dATP base points into a pocket containing two glutamine residues: Gln161 from one chain and Gln218 from the other. Of these Gln161 is conserved in T4NrdD and Gln218 is conservatively substituted to Glu. In T4NrdD these residues change conformation in detailed response to the H-bonding patterns presented by the effector base, inducing conformational changes in loop 2 that lead to altered specificity [29]. The conservation of Gln residues for effector recognition in evolutionarily distantly related RNRs, with their ability of the side chain to present H-bond donors or acceptors to the effector base by simple flipping, is interesting; however in tmNrdD neither of these critical residues H-bonds directly to the dATP base, suggesting that the mechanisms of allosteric specificity regulation in these distantly related class III RNRs are subtly different. A full analysis of the mechanism of allosteric specificity regulation will be presented elsewhere. Specificity effector binding involves a Mg2+ ion [29]. For T4NrdD the electron density was ambiguous and allowed the building of the effector phosphate groups in two possible conformations. In the present work we have identified the dNTP conformation unambiguously for tmNrdD by substituting Mn2+ for Mg2+ and collecting anomalous diffraction data (Fig 4A) at the Mn K edge. This places the metal ion in proximity to Gln210 and thus corrects the interpretation we made previously for T4NrdD [29], where the β-phosphate group was placed in this position. It cannot be excluded that the difference is species-specific, but we consider this possibility unlikely.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128199.g004: 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.
Mentions: The structure of tmNrdD has also been determined in complex with the allosteric substrate specificity effector dATP and its cognate substrate CTP (Fig 4), as well as with dATP alone. The effector dATP binds at the dimer interface, along the length of helices αA and αB of the α-β barrel (Figs 1B, 4A & 4B; the naming of secondary structure elements follows the convention for T4NrdD [9]). The dATP base points into a pocket containing two glutamine residues: Gln161 from one chain and Gln218 from the other. Of these Gln161 is conserved in T4NrdD and Gln218 is conservatively substituted to Glu. In T4NrdD these residues change conformation in detailed response to the H-bonding patterns presented by the effector base, inducing conformational changes in loop 2 that lead to altered specificity [29]. The conservation of Gln residues for effector recognition in evolutionarily distantly related RNRs, with their ability of the side chain to present H-bond donors or acceptors to the effector base by simple flipping, is interesting; however in tmNrdD neither of these critical residues H-bonds directly to the dATP base, suggesting that the mechanisms of allosteric specificity regulation in these distantly related class III RNRs are subtly different. A full analysis of the mechanism of allosteric specificity regulation will be presented elsewhere. Specificity effector binding involves a Mg2+ ion [29]. For T4NrdD the electron density was ambiguous and allowed the building of the effector phosphate groups in two possible conformations. In the present work we have identified the dNTP conformation unambiguously for tmNrdD by substituting Mn2+ for Mg2+ and collecting anomalous diffraction data (Fig 4A) at the Mn K edge. This places the metal ion in proximity to Gln210 and thus corrects the interpretation we made previously for T4NrdD [29], where the β-phosphate group was placed in this position. It cannot be excluded that the difference is species-specific, but we consider this possibility unlikely.

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