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The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site

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 genera...

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Autores principales: Aurelius, Oskar, Johansson, Renzo, Bågenholm, Viktoria, Lundin, Daniel, Tholander, Fredrik, Balhuizen, Alexander, Beck, Tobias, Sahlin, Margareta, Sjöberg, Britt-Marie, Mulliez, Etienne, Logan, Derek T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493059/
https://www.ncbi.nlm.nih.gov/pubmed/26147435
http://dx.doi.org/10.1371/journal.pone.0128199
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author Aurelius, Oskar
Johansson, Renzo
Bågenholm, Viktoria
Lundin, Daniel
Tholander, Fredrik
Balhuizen, Alexander
Beck, Tobias
Sahlin, Margareta
Sjöberg, Britt-Marie
Mulliez, Etienne
Logan, Derek T.
author_facet Aurelius, Oskar
Johansson, Renzo
Bågenholm, Viktoria
Lundin, Daniel
Tholander, Fredrik
Balhuizen, Alexander
Beck, Tobias
Sahlin, Margareta
Sjöberg, Britt-Marie
Mulliez, Etienne
Logan, Derek T.
author_sort Aurelius, Oskar
collection PubMed
description 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.
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spelling pubmed-44930592015-07-15 The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site Aurelius, Oskar Johansson, Renzo Bågenholm, Viktoria Lundin, Daniel Tholander, Fredrik Balhuizen, Alexander Beck, Tobias Sahlin, Margareta Sjöberg, Britt-Marie Mulliez, Etienne Logan, Derek T. PLoS One Research Article 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. Public Library of Science 2015-07-06 /pmc/articles/PMC4493059/ /pubmed/26147435 http://dx.doi.org/10.1371/journal.pone.0128199 Text en © 2015 Aurelius et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Aurelius, Oskar
Johansson, Renzo
Bågenholm, Viktoria
Lundin, Daniel
Tholander, Fredrik
Balhuizen, Alexander
Beck, Tobias
Sahlin, Margareta
Sjöberg, Britt-Marie
Mulliez, Etienne
Logan, Derek T.
The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
title The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
title_full The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
title_fullStr The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
title_full_unstemmed The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
title_short The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site
title_sort crystal structure of thermotoga maritima class iii ribonucleotide reductase lacks a radical cysteine pre-positioned in the active site
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493059/
https://www.ncbi.nlm.nih.gov/pubmed/26147435
http://dx.doi.org/10.1371/journal.pone.0128199
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