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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.


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.
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pone.0128199.g001: 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.

Mentions: Ribonucleotide reductases (RNRs) are highly important enzymes for all life, as they are solely responsible for the first committed step in the synthesis of the deoxyribonucleoside triphosphate (dNTP) building blocks of DNA [1]. Using three different types of radical chemistry they catalyze the reduction of either nucleoside diphosphates (NDP to dNDP) or nucleoside triphosphates (NTPs to dNTPs) [1]. RNRs are commonly divided into three main classes based on this radical chemistry, as well as their 3D structures, expression conditions and cofactor requirements: class I RNRs are dependent on a dinuclear metal cofactor and molecular O2 in almost all cases for generation of a stable tyrosyl radical [2]. The radical is then transferred to the active site through a 30–35 Å long proton-coupled electron transfer pathway terminating in a pair of Tyr residues in the active site of the reductase (Fig 1A). Class II RNRs, indifferent to oxygen levels, generate a 5’-deoxyadenosyl (5’-dAdo) radical through homolytic cleavage of the C-Co bond in their adenosylcobalamin cofactor [3,4]. Class III RNRs, strictly anaerobic, generate on a separate activating enzyme NrdG the same type of 5’-dAdo radical through homolytic cleavage of a C-S bond in S-adenosylmethionine, with the participation of a [4Fe-4S] cluster [5,6]. The 5’-dAdo radical in turn abstracts an H atom from a glycine residue in a C-terminal, inward-pointing loop in the reductase NrdD [7–9]. This glycyl radical is stable and can catalyze several dozen cycles before having to be regenerated [10,11].More than five decades of research on RNRs, including the crystal structure determination of several RNRs representing all three classes [9,12–14], have led to the insight that, despite often significant differences in sequence and radical generation mechanism, all RNRs are characterized by a 10-stranded α-β barrel fold containing at its heart the “finger loop” [12]. This loop harbors a critical, highly conserved cysteine residue responsible for initiation of the reduction reaction by abstraction of a hydrogen atom from the 3’ carbon of the substrate ribose ring [15]. The RNR fold is shared by other enzymes employing glycyl radicals, such as pyruvate formate lyase (PFL), glycerol dehydratase (GD) [16,17], 4-hydroxyphenylacetate decarboxylase (4-HPAD) [18] and benzylsuccinate synthase [19]. Two other active site cysteines on neighboring β-strands, conserved in class I and II RNRs, are responsible for electron and proton donation to the substrate during the reduction reaction and in the process form a disulphide bond that is indirectly reduced by thioredoxin (Trx) [20,21]. Only one of the latter two cysteines is conserved in class III RNR [9] and both reducing equivalents are provided by the small cosubstrate formate [22] in most systems characterized to date, although very recently Trx was also shown to be a possible reductant for the NrdD from Neisseria bacilliformis [23]. Nevertheless, whatever the means by which the stable or transient radicals described above are generated, it is thought that the single unifying factor of RNR radical chemistry is the transfer of these radicals to the critical cysteine at the initiation of catalysis [24] (Fig 1A). This cysteine is thought to be completely conserved across all RNRs, having been found in the sequences and structures of RNRs from all classes.


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) 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.
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Related In: Results  -  Collection

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pone.0128199.g001: 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.
Mentions: Ribonucleotide reductases (RNRs) are highly important enzymes for all life, as they are solely responsible for the first committed step in the synthesis of the deoxyribonucleoside triphosphate (dNTP) building blocks of DNA [1]. Using three different types of radical chemistry they catalyze the reduction of either nucleoside diphosphates (NDP to dNDP) or nucleoside triphosphates (NTPs to dNTPs) [1]. RNRs are commonly divided into three main classes based on this radical chemistry, as well as their 3D structures, expression conditions and cofactor requirements: class I RNRs are dependent on a dinuclear metal cofactor and molecular O2 in almost all cases for generation of a stable tyrosyl radical [2]. The radical is then transferred to the active site through a 30–35 Å long proton-coupled electron transfer pathway terminating in a pair of Tyr residues in the active site of the reductase (Fig 1A). Class II RNRs, indifferent to oxygen levels, generate a 5’-deoxyadenosyl (5’-dAdo) radical through homolytic cleavage of the C-Co bond in their adenosylcobalamin cofactor [3,4]. Class III RNRs, strictly anaerobic, generate on a separate activating enzyme NrdG the same type of 5’-dAdo radical through homolytic cleavage of a C-S bond in S-adenosylmethionine, with the participation of a [4Fe-4S] cluster [5,6]. The 5’-dAdo radical in turn abstracts an H atom from a glycine residue in a C-terminal, inward-pointing loop in the reductase NrdD [7–9]. This glycyl radical is stable and can catalyze several dozen cycles before having to be regenerated [10,11].More than five decades of research on RNRs, including the crystal structure determination of several RNRs representing all three classes [9,12–14], have led to the insight that, despite often significant differences in sequence and radical generation mechanism, all RNRs are characterized by a 10-stranded α-β barrel fold containing at its heart the “finger loop” [12]. This loop harbors a critical, highly conserved cysteine residue responsible for initiation of the reduction reaction by abstraction of a hydrogen atom from the 3’ carbon of the substrate ribose ring [15]. The RNR fold is shared by other enzymes employing glycyl radicals, such as pyruvate formate lyase (PFL), glycerol dehydratase (GD) [16,17], 4-hydroxyphenylacetate decarboxylase (4-HPAD) [18] and benzylsuccinate synthase [19]. Two other active site cysteines on neighboring β-strands, conserved in class I and II RNRs, are responsible for electron and proton donation to the substrate during the reduction reaction and in the process form a disulphide bond that is indirectly reduced by thioredoxin (Trx) [20,21]. Only one of the latter two cysteines is conserved in class III RNR [9] and both reducing equivalents are provided by the small cosubstrate formate [22] in most systems characterized to date, although very recently Trx was also shown to be a possible reductant for the NrdD from Neisseria bacilliformis [23]. Nevertheless, whatever the means by which the stable or transient radicals described above are generated, it is thought that the single unifying factor of RNR radical chemistry is the transfer of these radicals to the critical cysteine at the initiation of catalysis [24] (Fig 1A). This cysteine is thought to be completely conserved across all RNRs, having been found in the sequences and structures of RNRs from all classes.

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.