Ribonucleotide reductases: Substrate specificity by allostery

doi:10.1016/j.bbrc.2010.02.108

Biochemical and Biophysical Research Communications

Volume 396, Issue 1, 21 May 2010, Pages 19-23

https://doi.org/10.1016/j.bbrc.2010.02.108 Get rights and content

Abstract

Ribonucleotide reductases catalyze in all living organisms the production of deoxynucleotides from ribonucleotides. A single enzyme provides a balanced supply of the four dNTPs required for DNA replication. Three different but related classes of enzymes are known. Each class catalyzes the same chemistry using a common radical mechanism involving a thiyl radical of the enzyme but the three classes employ different mechanisms for the generation of the radical. For each class a common allosteric mechanism with ATP and dNTPs as effectors directs the substrate specificity of the enzymes ensuring the appropriate balance of the four dNTPs for DNA replication. Recent crystallographic studies of the catalytic subunits from each class in combination with allosteric effectors, with and without cognate substrates, delineated the structural changes caused by effector binding that direct the specificity of the enzymes towards reduction of the appropriate substrate.

Section snippets

Ribonucleotide reductases in the RNA world

Life, as we know it, requires two fundamentals: self-replication and catalysis. Today these are provided by three separate macromolecules: DNA takes care of replication, protein of catalysis and RNA occupies an intermediate position between DNA and protein. Early during evolution life must have started with only one of them, with a simpler chemical structure than today, but already with the dual ability for self-replication and catalysis. Only RNA fits these requirements and according to a

All ribonucleotide reductases are radical enzymes

Ribonucleotide reduction is catalyzed by a complicated and highly sophisticated system. Today, life is not possible without it. During the bicentennial festivities for the birth of the Karolinska Institute we can in 2010 also celebrate the 50th anniversary of the discovery of ribonucleotide reductases at the Karolinska Institute. The first reductase appeared in an extract from Escherichia coli[5]. Together with an enthusiastic group of young co-workers, primarily Agne Larsson, Lars Thelander

Generation of the protein radical

Whereas the activation of ribose by a thiyl radical for the removal of the OH-group is identical for all enzymes, major differences appear in the way in which this radical is generated. On the basis of these differences the enzymes are divided into three classes [2], each with a different metallo-cofactor for radical generation (Fig. 1). Class I reductases exist only in aerobic eukaryotes and prokaryotes. In E. coli the enzyme is a complex of two non-identical protein dimers, named R1 and R2.

The evolution of ribonucleotide reduction

How did ribonucleotide reduction evolve during the transition of the RNA to the DNA world [13], [14], [15]? Did each of the three classes evolve independently, or do they have a common origin and then evolved further by divergent evolution? And if so, which of them came first, or rather which of them is the closest relative of a more primitive common ancestor? In sequence comparisons, one finds a limited global homology between class I and II that does not include class III. The overall

Activity and specificity of ribonucleotide reductases are governed by allostery

DNA replication requires a balanced supply of all the four dNTPs. Disturbances result in increased mutation rates and may lead to disease. The dNTPs are provided by a single enzyme and their balance is achieved by the enzyme’s complicated allosteric regulation. When in the early 1960s we found that ATP was required for the reduction of CDP we did not realize that we were looking at an allosteric effect nor did we understand that different allosteric effectors are required for the reduction of

Substrate specificity of the E. coli enzyme

Allosteric regulation typically is recognized from sigmoidal substrate binding curves of oligomeric proteins [17], [18]. The allosteric effector changes subunit interactions affecting the binding of substrate and enzyme activity. In our case an effect of this kind may cause the effects of ATP and dATP at the activity site. It does, however, not explain changes in substrate specificity caused by effector binding at the specificity site. We proposed instead that in this case effector binding

Substrate specificity of the Thermotoga maritima reductase

A crystallographic investigation of the ribonucleotide reductase from T. maritima, a hyperthermophilic class II enzyme, showed the detailed structural changes responsible for the regulation of substrate specificity [2], [24]. The enzyme is a dimeric protein that employs adenosyl cobalamin to generate the thiyl radical for the activation of the ribotide. In the structure, the substrate is bound as in the E. coli protein suggesting a similar final catalytic mechanism. Also the allosteric

Generality of allosteric mechanism

Many of the key amino acid residues involved in nucleotide binding are conserved not only in most class II enzymes but also in class I reductases providing evidence that the rules discovered for the Thermotoga reductase can be generally applied to reductases from the two classes. Among the eukaryotic enzymes only the catalytic subunit of yeast has been investigated by crystallography with similar results as the Thermotoga reductases confirming that loop 2 also in a eukaryotic class I enzyme is

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Citation Excerpt :
First, allosteric regulation controls RNR overall activity and substrate specificity. While the allosteric specificity site regulates the balance among the four dNTP pools, the allosteric activity site controls the total dNTP pool size by monitoring the dATP/ATP ratio (reviewed in [12]). In response to DNA damage or DNA replication stress, the Mec1-Tel1/Rad53/Dun1 checkpoint kinase cascade promotes yeast RNR activity.
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Ribonucleotide reductases: Substrate specificity by allostery

Abstract

Section snippets

Ribonucleotide reductases in the RNA world

All ribonucleotide reductases are radical enzymes

Generation of the protein radical

The evolution of ribonucleotide reduction

Activity and specificity of ribonucleotide reductases are governed by allostery

Substrate specificity of the E. coli enzyme

Substrate specificity of the Thermotoga maritima reductase

Generality of allosteric mechanism

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

Prog. Biophys. Mol. Biol.

J. Biol. Chem.

Prog. Nucleic Acid Res. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Structure

Structure