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.


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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128199.g003: 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.

Mentions: The sequence that multiple sequence alignment programs typically align with the tip of the finger loop in the absence of structural guidance, 328SCCR331 (288MGCR291 in T4) is found in an extended loop region on the far side of the α-β barrel from the active site (Fig 3). The structure becomes disordered immediately after the first cysteine in the SCCR motif, Cys329, and the rest of the loop has the sequence characteristics of an intrinsically disordered region.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128199.g003: 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.
Mentions: The sequence that multiple sequence alignment programs typically align with the tip of the finger loop in the absence of structural guidance, 328SCCR331 (288MGCR291 in T4) is found in an extended loop region on the far side of the α-β barrel from the active site (Fig 3). The structure becomes disordered immediately after the first cysteine in the SCCR motif, Cys329, and the rest of the loop has the sequence characteristics of an intrinsically disordered region.

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.