Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity DOI
Andrew J. Knappenberger, Md. Faiz Ahmad, Rajesh Viswanathan

et al.

Biochemistry, Journal Year: 2016, Volume and Issue: 55(41), P. 5884 - 5896

Published: Sept. 16, 2016

Class I ribonucleotide reductase (RR) maintains balanced pools of deoxyribonucleotide substrates for DNA replication by converting ribonucleoside diphosphates (NDPs) to 2'-deoxyribonucleoside (dNDPs). Binding deoxynucleoside triphosphate (dNTP) effectors (ATP/dATP, dGTP, and dTTP) modulates the specificity class RR CDP, UDP, ADP, GDP substrates. Crystal structures bacterial eukaryotic RRs show that dNTP NDP bind on either side a flexible nine-amino acid loop (loop 2). Interactions with effector nucleobase alter 2 geometry, resulting in changes among four RR. However, functional groups proposed drive remain untested. Here, we use analogue triphosphates determine human (hRR) specificity. The results demonstrate 5-methyl, O4, N3 dTTP contribute GDP. O6 protonated N1 dGTP direct ADP. In contrast, unprotonated adenosine is primary determinant ATP/dATP-directed CDP. Structural models from X-ray crystallography suggest chain D287 involved binding dTTP, but not dATP/ATP. This feature consistent experimental showing D287A mutant hRR deficient allosteric regulation ATP/dATP. Together, these data define are drivers provide constraints evaluating regulation.

Language: Английский

X-ray Scattering Studies of Protein Structural Dynamics DOI
Steve P. Meisburger, William C. Thomas, Maxwell B. Watkins

et al.

Chemical Reviews, Journal Year: 2017, Volume and Issue: 117(12), P. 7615 - 7672

Published: May 30, 2017

X-ray scattering is uniquely suited to the study of disordered systems and thus has potential provide insight into dynamic processes where diffraction methods fail. In particular, while crystallography been a staple structural biology for more than half century will continue remain so, major limitation this technique lack information. Solution become an invaluable tool in mechanistic studies biological macromolecules large conformational changes are involved. Such include allosteric enzymes that play key roles directing metabolic fluxes biochemical pathways, as well large, assembly-line type synthesize secondary metabolites with pharmaceutical applications. Furthermore, information on protein dynamics via diffuse patterns overlaid Bragg diffraction. Historically, these have very difficult interpret, but recent advances detection led renewed interest analysis way probe correlated motions. Here, we review theory highlight scattering-based investigations solutions crystals, particular focus complex enzymes.

Language: Английский

Citations

95
NrdR in Streptococcus and Listeria spp.: DNA Helix Phase Dependence of the Bacterial Ribonucleotide Reductase Repressor DOI Creative Commons
Saher Shahid, Mateusz Bałka, Daniel Lundin

et al.

Molecular Microbiology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 18, 2025

ABSTRACT NrdR is a universal transcriptional repressor of bacterial genes coding for ribonucleotide reductases (RNRs), essential enzymes that provide DNA building blocks in all living cells. Despite its prevalence, the mechanism has been scarcely studied. We report biochemical, biophysical, and bioinformatical characterization binding sites from two major pathogens phylum Bacillota Listeria monocytogenes Streptococcus pneumoniae . consists Zn‐ribbon domain followed by an ATP‐cone domain. show it forms tetramers bind to when loaded with ATP dATP, but if only ATP, various oligomeric complexes unable DNA. The DNA‐binding site L. pair boxes separated 15–16 bp, whereas S. , are unusually long spacers 25–26 bp. This observation triggered comprehensive study four NrdRs Escherichia coli Streptomyces coelicolor series dsDNA fragments where were 12–27 vitro results confirmed vivo E. revealed binds most efficiently there integer number turns between center boxes. facilitates prediction genomes suggests conserved throughout It sheds light on RNR regulation since does not occur eukaryotes, opens way development novel antibiotics.

Language: Английский

Citations

1
The Origin and Evolution of Ribonucleotide Reduction DOI Creative Commons
Daniel Lundin, Gustav Berggren, Derek T. Logan

et al.

Life, Journal Year: 2015, Volume and Issue: 5(1), P. 604 - 636

Published: Feb. 27, 2015

Ribonucleotide reduction is the only pathway for de novo synthesis of deoxyribonucleotides in extant organisms. This chemically demanding reaction, which proceeds via a carbon-centered free radical, catalyzed by ribonucleotide reductase (RNR). The mechanism has been deemed unlikely to be ribozyme, creating an enigma regarding how building blocks DNA were synthesized at transition from RNA- DNA-encoded genomes. While it entirely possible that different was later replaced with modern mechanism, here we explore evolutionary and biochemical limits origin RNA + protein world suggest model prototypical (protoRNR). From protoRNR evolved ancestor RNRs, urRNR, diversified into three classes. Since initial radical generation differs between classes, difficult establish generated urRNR. Here similar B12-dependent class II RNRs.

Language: Английский

Citations

91
Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer DOI Creative Commons
Nozomi Ando, Haoran Li, Edward J. Brignole

et al.

Biochemistry, Journal Year: 2015, Volume and Issue: 55(2), P. 373 - 381

Published: Dec. 21, 2015

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion ribonucleotides to deoxyribonucleotides. Because its essential role cell division, human RNR is a target number anticancer drugs clinical use. Like other class Ia RNRs, requires both radical-generation subunit (β) and nucleotide-binding (α) activity. their complex dependence on allosteric effectors, however, active inactive quaternary forms many RNRs have remained question. Here, we present an X-ray crystal structure α presence inhibiting levels dATP, depicting ring-shaped hexamer (α6) where sites line inner hole. Surprisingly, our small-angle scattering (SAXS) results indicate that similar ATP, activating effector. In cases, α6 assembled from dimers (α2) without previously proposed tetramer intermediate (α4). However, show with SAXS electron microscopy at millimolar ATP-induced can further interconvert higher-order filaments. Differences dATP- were examined β activity assays as function ATP or dATP. Together, these suggest dATP-induced more stable than stabilization this configuration provides mechanism prevent access site α.

Language: Английский

Citations

58
Novel ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit DOI Creative Commons
Inna Rozman Grinberg, Daniel Lundin, Mahmudul Hasan

et al.

eLife, Journal Year: 2018, Volume and Issue: 7

Published: Feb. 1, 2018

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, ATP-cone, has been found exclusively catalytic subunit. two class I RNR subclasses whose subunit lacks we discovered ATP-cones radical-generating The ATP-cone Leeuwenhoekiella blandensis regulates via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP non-productive tetramers, resulting different holoenzymes. tetramer forms interactions between ATP-cones, shown a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. summary, lack domain was compensated transfer to To our knowledge, this represents first observation components same enzyme complex.

Language: Английский

Citations

53
Radicals in Biology: Your Life Is in Their Hands DOI
JoAnne Stubbe, Daniel G. Nocera

Journal of the American Chemical Society, Journal Year: 2021, Volume and Issue: 143(34), P. 13463 - 13472

Published: Aug. 23, 2021

Radicals in biology, once thought to all be bad actors, are now known play a central role many enzymatic reactions. Of the radical-based enzymes, ribonucleotide reductases (RNRs) pre-eminent as they essential biology of organisms by providing building blocks and controlling fidelity DNA replication repair. Intense examination RNRs has led development new tools guiding framework for study radicals pointing way future frontiers radical enzymology.

Language: Английский

Citations

39
Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones DOI Creative Commons

Renzo Johansson,

Venkateswara Rao Jonna, Rohit Kumar

et al.

Structure, Journal Year: 2016, Volume and Issue: 24(6), P. 906 - 917

Published: April 30, 2016

Language: Английский

Citations

36
Convergent allostery in ribonucleotide reductase DOI Creative Commons
William C. Thomas, F. Phil Brooks, Audrey A. Burnim

et al.

Nature Communications, Journal Year: 2019, Volume and Issue: 10(1)

Published: June 14, 2019

Abstract Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides deoxyribonucleotides. Within RNR family, class Ib RNRs are notable for being largely restricted bacteria, including many pathogens, and lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report emergence distinct unexpected activity regulation in sole model organism Bacillus subtilis . Using hypothesis-driven structural approach combines strengths small-angle X-ray scattering (SAXS), crystallography, cryo-electron microscopy (cryo-EM), describe reversible interconversion six unique structures, flexible active tetramer two inhibited helical filaments. These structures reveal conformational gymnastics necessary molecular basis its control via convergent form allostery.

Language: Английский

Citations

35
Bacterial transcriptional repressor NrdR – a flexible multifactorial nucleotide sensor DOI Creative Commons
Inna Rozman Grinberg, Ornella Bimaï, Saher Shahid

et al.

FEBS Journal, Journal Year: 2025, Volume and Issue: unknown

Published: March 3, 2025

NrdR is a bacterial transcriptional repressor consisting of zinc (Zn)‐ribbon domain followed by an ATP‐cone domain. Understanding its mechanism action could aid the design novel antibacterials. binds specifically to two “NrdR boxes” upstream ribonucleotide reductase operons, which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, in last we identified new box. We show that E. (EcoNrdR) similar binding strength all three sites when loaded with ATP plus deoxyadenosine triphosphate (dATP) or equivalent diphosphate combinations. No other combination adenine nucleotides promotes DNA. present crystal structures EcoNrdR–ATP–dATP EcoNrdR–ADP–dATP, are first high‐resolution NrdR. have also determined cryo‐electron microscopy DNA‐bound filaments EcoNrdR–ATP. Tetrameric forms EcoNrdR involve alternating interactions between pairs Zn‐ribbon domains ATP‐cones. The reveal considerable flexibility relative orientation ATP‐cones vs domains. structure shows significant conformational rearrangements Zn‐ribbons accompany DNA while retain same orientation. In contrast, ATP‐loaded sequester DNA‐binding residues such they unable bind Our results, previous structural biochemical study, point highly flexible that, correct nucleotides, adapt optimal promoter‐binding conformation.

Language: Английский

Citations

0
Analysis of insertions and extensions in the functional evolution of the ribonucleotide reductase family DOI Creative Commons
Audrey A. Burnim, Da Xu, Matthew A. Spence

et al.

Protein Science, Journal Year: 2022, Volume and Issue: 31(12)

Published: Oct. 29, 2022

Abstract Ribonucleotide reductases (RNRs) are used by all free‐living organisms and many viruses to catalyze an essential step in the de novo biosynthesis of DNA precursors. RNRs remarkably diverse primary sequence cofactor requirement, while sharing a conserved fold radical‐based mechanism for nucleotide reduction. In this work, we expand on our recent phylogenetic inference entire RNR family describe evolutionarily relatedness insertions extensions around structurally homologous catalytic barrel. Using evo‐velocity similarity network (SSN) analyses, show that N‐terminal regulatory motif known as ATP‐cone domain was likely inherited from ancestral RNR. By combining SSN analysis with AlphaFold2 predictions, also C‐terminal class II can contain folded domains share homology Fe‐S cluster assembly protein. Finally, using AlphaFold2, catalytically insertion finger loop is tightly coupled mechanism. Based these results, propose evolutionary model diversification family.

Language: Английский

Citations

14