Ribonucleotide Reductase

Introduction

Ribonucleotide reductase, , is one of the major enzymes that aids in the synthesis of deoxyribonucleotides. These precursors of DNA are vital for DNA synthesis, so RNRs are required in all living cells to replicate and repair DNA. The reaction catalyzed through RNR is the only biochemical pathway that can synthesize new deoxyribonucleotide triphosphates (dNTPs).^[1] This enzyme has led to and contributed to the evolution of genetic material that exists today. RNR consists of three different classes: I, II, and III. The classes differ in which they require different metal cofactors in order to initiate the reaction. This then leads to different environmental factors affecting the enzyme and its different classes.

Structure

The structure of ribonucleotide reductase is composed of two components. One portion is the radical generator which produces and stores the radical. This portion is used to oxidize the substrate to its radical form which is the first step of the overall reaction. The second portion of the structure consists of a reductase. The reductase is the same for all three classes, but the radical generator differs. ^[2] Class I ribonucleotide reductase produces the stable radical, , through the dinuclear or of the subunit. The three different classes of this enzyme have a cysteine residue that is located at the protein loop of the active site. Class II enzymes differ in which it uses cobalamin as a cofactor for the radical. The protein loop is in the center of the alpha, beta-barrel of the structural motif. The cysteine residue is conserved until it is converted to a thiyl radical. The conversion to the thiyl radical is needed to initiate substrate turnover. The different classes of RNR are different but do show some similarities which suggest that they all evolved from one common reductase. ^[3] Class III of RNR is comprised of two proteins that require an iron-sulfur center.

Function

The conversion from ribonucleotides to deoxyribonucleotides is a radically-based reaction. Organic free radicals are stored by RNR until they are needed for catalysis. Class I is the most studied and most well known enzyme that is a part of the RNR family. It is composed of two subunits, alpha and beta, that are homodimeric. The alpha subunit contains the active site and two allosteric sites. The active site contains a catalytic subunit, and the allosteric sites are used for regulation, enzyme activity, and substrate specificity. The beta portion contains the metal cofactor that initiates reduction. Class I is subdivided into three more enzymes that are class Ia, Ib, and Ic. Class one is split even further because it is depending on the specific type of metal center that is needed for protein radical. All three classes share the same function for the beta subunit which is synthesizing the radical which is transferred using a radical transfer pathway. Class Ia requires a di-iron center as the nrdAB gene codes for this enzyme. Class Ib uses either a di-manganese center or a di-ferric center. This specific class differs from the other class I enzymes by lacking the active site located at the N-terminal portion of the protein. This enzyme is coded by the gene nrdHIEF Class Ic is encoded by the nrdAB gene, and it requires a manganese-iron center. Class I and its subdivision are all aerobic and require oxygen for the generation of the radical. This enzyme can be found in eukaryotes, archaea, eubacteria, and bacteriophages. Class II is encoded by the nrdj gene that harbors the allosteric sites and active site of the enzyme, so the enzyme itself only contains one subunit which is alpha or alpha2. There is an allosteric specificity site, but no allosteric active site. S-adenosylcobalamin is used as a cofactor in order to generate the cysteinyl radical. ^[1] The radical is formed when the bond between the adenosyl and cobalamin is cleaved. It has been recently discovered that several class II RNRs with catalytic domains of the B12-dependent enzyme are related to class I and III RNRs. The reaction for class II can be aerobic or anaerobic because it is oxygen independent. This class is found in archaea, eubacteria, and bacteriophages. It is most studied in microorganisms such as Lactobacillus leichmannii. Class III is composed of two proteins that are homodimeric. It is encoded by nrdG and nrdD genes which contain a large catalytic subunit with an active site and two allosteric sites used for regulation. The NrdG protein, activase, initiates the generation of the radical which requires binding of the iron-sulfur cluster in the center of the protein with S-adenosylmethionine. The interaction between the metal center and adenosylmethionine synthesizes a glycyl radical which causes this reaction to be completely anaerobic. The glycyl radical forms at the C-terminal of the protein which is sensitive to oxygen. ^[1] Along with that and the potential of the metal center oxidizing, this reaction cannot run in the presence of oxygen. This class is found in bacteriophages, eubacteria, and archaea.

Disease

Activity of RNR is controlled by substrate specificity and enzymatic activity. Substrate activity functions by allowing the binding of different nucleotides which results in the reduction of a specific NTP. This regulation occurs at the active site. ATP or dATP can bind to activate or inhibit activity which allows for enzymatic activity to be controlled. Mutations can occur within the genome if there is an imbalance in the pools of dNTPs. These are the building blocks for DNA, and if the pool is altered, then it can lead to genomic instability. ^[4] Having accurate DNA repair and replication allows for development, tumor-free cells, and growth. Having issues within the genome can cause several human diseases, mitochondrial disorders, and cause a person to be more susceptible to infection and cancer. High expressions of RNR is a main characteristic for cancers since it influences DNA replication so heavily. For this reason, RNRs play a major role in targeting anticancer and antibacterial drugs and therapies. RNR inhibitors are being studied to see if they can potentially serve as cancer treatment that is effective. More and more research is being done everyday in order to fully understand ribonucleotide reductase and all of its classes. For decades, dNTP machinery and RNR has been used and exploited for therapeutic benefits. They serve an important target for developing an efficient cancer drug.