Ribonucleotide Reductase - Structure

Structure

The iron-dependent enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis. Class I RNR enzymes are constructed from large RNR1 and small RNR2 subunits which associate to form an active heterodimeric tetramer. Since the enzyme catalyses the de novo synthesis of deoxyribonucleotides (dNTPs), precursors to DNA synthesis, it is essential for cell proliferation.

In humans, the RNR1 subunit is encoded by the RRM1 gene while there are two isoforms of the RNR2 subunit, encoded by the RRM2 and RRM2B genes:

Each RNR1 monomer consists of three domains:

• one mainly helical domain comprising the 220 N-terminal residues,
• a second large ten-stranded α/β structure comprising 480 residues,
• and a third small five-stranded α/β structure comprising 70 residues.

In Pfam, the second domain has been interpreted as two separate domains:

• a shorter all-alpha N-terminal domain,
• and a longer barrel C-terminal domain.

RNR2 contains a diferric iron center and a stable tyrosyl radical. In E. coli, the tyrosyl radical is located at position 122 (Y122) providing the stable radical for the Class I RNR2 subunits. In A. aegypti, this tyrosyl radical is located at position 184 (Y184). The tyrosyl radical is deeply buried inside the protein in a hydrophobic environment, located close to the iron center that is used in the stabilization of a tyrosyl radical. The structure of two μ-oxo-linked irons is dominated by ligands that serve as iron binding sites: four carboxylates and two histidines (H180 and H277). Association occurs between the C-terminus of RNR2 and the C-terminus of RNR1. Enzymatic activity is dependent on association of the RNR1 and RNR2 subunits. The active site consists of the active dithiol groups from the RNR1 as well as the diferric center and the tyrosyl radical from the RNR2 subunit.

Other residues of RNR2, such as aspartate (D273), tryptophan (W48), and tyrosine (Y356) further stabilize the active-site tyrosyl radical thus allowing electron transfer. These residues help in the transfer of the radical electron from tyrosine (Y122) of RNR2 to cysteine (C439) of RNR1. The electron transfer begins on RNR2 tyrosine (Y122) and continues in RNR2 to tryptophan (W48), which is separated from RNR1 tyrosine (Y731) by 2.5 nanometers. Electron transfer from RNR2 to RNR1 occurs via tyrosine (Y356 to Y731) and continues on through tyrosine (Y730) to cysteine (C439) in the active site. Site-directed mutations of the RNR primary structure indicate that all residues cited above participate in the long distance transfer of the free radical to the active site.

In A. aegypti mosquitoes, RNR1 retains most of the crucial amino acid residues, including aspartate (D64) and valine (V292 or V284), that are necessary in allosteric regulation; proline (P210 and P610), leucine (L453 and L473), and methionine (M603) residues that are located in the hydrophobic active site; cysteine (C225, C436 and C451) residues that are involved in removal of a hydrogen atom and transfer of the radical electron at the active site; cysteine (C225 and C436), asparagine (N434), and glutamate (E441) residues that bind the ribonucleotide substrate; tyrosine (Y723 and Y743) residues that dictate the radical transfer; and cysteine (C838 and C841) residues that are used in the regeneration of dithiol groups in the active site.