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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.


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.
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pone.0128199.g007: 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.

Mentions: To understand how widespread this class of RNR might be, we performed sequence analysis using hidden Markov models from RNRdb2 (http://rnrdb.pfitmap.org). In RNRdb2, the highly diverse RNR protein family is divided into subclasses based on phylogenetic evidence [43]. Subclass NrdDh, to which tmNrdD belongs, possesses a hydrophobic residue equivalent to Ile359. The organization of NrdDh is exemplified by the tmNrdD structure, and the subclass can be further subdivided into four groups based on sequence similarity: NrdDh1-4 (Fig 7 and S6 Fig). For comparison, the T4NrdD structure belongs to subclass IIIc (see RNRdb2). NrdDh is encoded and evolutionarily conserved in genomes with a phylogenetically wide distribution–two proteobacterial classes, Firmicutes and Bacteroidetes in addition to Thermotogae. The Ile at position 359 in tmNrdD is shared by all NrdDh1 sequences, all from Thermotogales except two from Clostridium phages, but is Leu in NrdDh2 and -3, and Phe in NrdDh4 (Fig 7 and S6 Fig), although an insert prior to the Ile in the NrdDh1 sequences creates some uncertainty as to exactly which residue is homologous to the tmNrdDh Ile in the other three groups. Another characteristic of the NrdDh1 group is a Tyr at position 227, which replaces the Phe conserved in other NrdDh as well as all other NrdDs. All other NrdDh sequences identified by HMMER using specific profiles for NrdDh possess the SCCR motif–except NrdDh4 which has a Met instead of Ser–in combination with the putative finger loop motif containing either Leu or Phe instead of Ile (Fig 7 and S6 Fig). We have not found this motif outside of NrdDh. The SCCR motif is sandwiched in between other well defined, conserved sequence motifs defining strands βE and βF, and we predict that the SCCR sequence may be on the far side of the barrel in these structures as well.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128199.g007: 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.
Mentions: To understand how widespread this class of RNR might be, we performed sequence analysis using hidden Markov models from RNRdb2 (http://rnrdb.pfitmap.org). In RNRdb2, the highly diverse RNR protein family is divided into subclasses based on phylogenetic evidence [43]. Subclass NrdDh, to which tmNrdD belongs, possesses a hydrophobic residue equivalent to Ile359. The organization of NrdDh is exemplified by the tmNrdD structure, and the subclass can be further subdivided into four groups based on sequence similarity: NrdDh1-4 (Fig 7 and S6 Fig). For comparison, the T4NrdD structure belongs to subclass IIIc (see RNRdb2). NrdDh is encoded and evolutionarily conserved in genomes with a phylogenetically wide distribution–two proteobacterial classes, Firmicutes and Bacteroidetes in addition to Thermotogae. The Ile at position 359 in tmNrdD is shared by all NrdDh1 sequences, all from Thermotogales except two from Clostridium phages, but is Leu in NrdDh2 and -3, and Phe in NrdDh4 (Fig 7 and S6 Fig), although an insert prior to the Ile in the NrdDh1 sequences creates some uncertainty as to exactly which residue is homologous to the tmNrdDh Ile in the other three groups. Another characteristic of the NrdDh1 group is a Tyr at position 227, which replaces the Phe conserved in other NrdDh as well as all other NrdDs. All other NrdDh sequences identified by HMMER using specific profiles for NrdDh possess the SCCR motif–except NrdDh4 which has a Met instead of Ser–in combination with the putative finger loop motif containing either Leu or Phe instead of Ile (Fig 7 and S6 Fig). We have not found this motif outside of NrdDh. The SCCR motif is sandwiched in between other well defined, conserved sequence motifs defining strands βE and βF, and we predict that the SCCR sequence may be on the far side of the barrel in these structures as well.

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.