Mouse ribonucleotide reductase: from genes to proteins

Abstract

903Ribonucleotide reductase (EC 1.17.4.1 -2) catalyses the direct reduction of ribonucleotides to the corresponding deoxyribonucleotides. This reaction enabled the evolution from the early 'RNA world' to the present 'DNA world'. Storage of genetic information as DNA instead of RNA is much more stable and this was a prerequisite for the later development of multiple, separate, species [l].Ribonucleotide reduction is a chemically difficult reaction and requires free radical chemistry. We now know of three different classes of ribonucleotide reductases [ 11. Catalysis in all three classes is dependent on an enzyme-centred free radical, which in the class I enzymes is an iron-centre-generated tyrosyl free radical. Class I1 uses adenosyl cobalamin as a cofactor, which generates a transient radical during catalysis. Finally, class I11 uses a glycine radical which is generated in a reaction that requires S-adenosylmethionine.All plant and eukaryotic ribonucleotide reductases described so far belong to class I. Here the enzyme consists of two non-identical homodimeric subunits, proteins R1 and R2. This is very similar to the extensively studied Escherichiu coli nrd AB enzyme [2-41. The 2 x 90 kDa mouse R1 protein contains the ribonucleoside diphosphate substrate binding sites as well as two different types of nucleoside triphosphate effector binding sites. It also contains a number of redox active disulphides that participate in electron transfer from NADPH/thioredoxin/ glutaredoxin. There is only one enzyme catalysing the reduction of all four different ribonucleotides, but both the overall activity and the substrate specificity are controlled by allosteric regulation. This control ensures a balanced supply of the four deoxyribonucleotides during DNA replication and repair to prevent misincorporation and mutagenesis [3,5].Each 45 kDa polypeptide of the mouse R2 protein contains a binuclear iron centre which, during its formation, generates a stable tyrosyl free radical that is essential for activity [6,7]. The iron centre was shown to be labile at physiological temperatures, resulting in a loss of about *To whom correspondence should be addressed. 50% of the iron after 30 min at 37°C [8]. Therefore there is a continuous regeneration of the iron-tyrosyl radical centre in mammalian R2 in vivo. The regeneration requires ferrous iron and oxygen and explains why mammalian ribonucleotide reductase activity, and consequently also DNA synthesis and cell growth, is sensitive to iron chelators and oxygen deprivation. This was demonstrated in direct EPR measurements of the R2 tyrosyl radical in intact cells treated with iron chelators [8]. The lability of the iron centre in the mammalian R2 protein is in marked contrast to the E. coli R2 protein, which does not easily lose its iron.The crystal structures of the E. coli R1 and R2 proteins show that the tyrosyl free radical is buried lOA (1 nm) away from the surface of the very rigid R2 protein [9]. Furthermore, the active site of the R1 protein is located 25A from the most likely R...

The role of tumor metastasis suppressors in cancers of breast and prostate

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Ribonucleotide reductase M1 gene promoter activity, polymorphisms, population frequencies, and clinical relevance

“…Ribonucleotide reductase catalyses the rate limiting step of deoxyribonucleotide formation [1][2][3]. It is the only enzyme in human cells capable of such synthesis and thus crucially important in DNA synthesis and repair.…”

Evidence by Mutagenesis that Tyr370 of the Mouse Ribonucleotide Reductase R2 Protein Is the Connecting Link in the Intersubunit Radical Transfer Pathway

“…NMR studies showed that the mouse R2 protein in solution contains ϳ30 highly flexible residues, most of which can be attributed to the C terminus of the polypeptide chain (20). The C terminus is required for complex formation between the R1 and R2 proteins (21)(22)(23), and upon binding to the R1 protein, the mobile R2 protein tail becomes rigid (20). Peptides and peptidomimetics corresponding to the R2 protein C terminus specifically inhibit ribonucleotide reductase (24 -27).…”