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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

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.
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pone.0128199.g002: 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.

Mentions: The tmNrdD structure contains a C-terminal loop harbouring the glycyl radical site (Gly621), which dips into the α-β barrel such that its tip meets that of the finger loop in the active site (Fig 1D). The electron density for both loops is very well defined (Fig 2A). Since the T4 enzyme structure was solved as an oxygen-insensitive Gly->Ala mutant [9], the present structure allows the native conformation of the radical Gly in an anaerobic ribonucleotide reductase to be observed in its resting state prior to activation, as also seen by Wei et al. [23]. The flanking sequence and conformation around Gly621 are very similar to those in the G580A mutant of T4NrdD, as well as PFL [32], GD [17] and 4-HPAD [18] (Fig 2B). Gly621 itself is completely buried from solvent and presumably undergoes a significant conformational change in order for its hydrogen atom to be abstracted by the 5’-dAdo radical generated by the activase, as postulated previously for T4NrdD [9] and PFL [33]. This is almost certainly linked to conformational changes in the entire C-terminal domain, which includes the Zn-binding domain, but comparison with the T4NrdD structure does not allow us to speculate whether the different orientations of the Zn domain are due to an inherent flexibility or entirely due to species variation.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128199.g002: 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.
Mentions: The tmNrdD structure contains a C-terminal loop harbouring the glycyl radical site (Gly621), which dips into the α-β barrel such that its tip meets that of the finger loop in the active site (Fig 1D). The electron density for both loops is very well defined (Fig 2A). Since the T4 enzyme structure was solved as an oxygen-insensitive Gly->Ala mutant [9], the present structure allows the native conformation of the radical Gly in an anaerobic ribonucleotide reductase to be observed in its resting state prior to activation, as also seen by Wei et al. [23]. The flanking sequence and conformation around Gly621 are very similar to those in the G580A mutant of T4NrdD, as well as PFL [32], GD [17] and 4-HPAD [18] (Fig 2B). Gly621 itself is completely buried from solvent and presumably undergoes a significant conformational change in order for its hydrogen atom to be abstracted by the 5’-dAdo radical generated by the activase, as postulated previously for T4NrdD [9] and PFL [33]. This is almost certainly linked to conformational changes in the entire C-terminal domain, which includes the Zn-binding domain, but comparison with the T4NrdD structure does not allow us to speculate whether the different orientations of the Zn domain are due to an inherent flexibility or entirely due to species variation.

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