REVIEW Susana Pereira Æ Nuno M.F.S.A. Cerqueira Pedro Alexandrino Fernandes Æ Maria Joa˜o Ramos Computational studies on class I ribonucleotide reductase: understanding the mechanisms of action and inhibition of a cornerstone enzyme for the treatment of cancer Received: 21 May 2005 / Revised: 21 September 2005 / Accepted: 28 September 2005 / Published online: 29 October 2005 Ó EBSA 2005 Abstract This review provides a synthesis of recent work, using computational methods, on the action and inhi- bition mechanisms of class I ribonucleotide reductase (RNR). This enzyme catalyzes the rate-limiting step of the pathway for the synthesis of DNA monomers and, therefore, has long been regarded as an important target for therapies aiming to control pathologies that depend strongly on DNA replication. In fact, over the last years, several molecules, which are able to impair RNR activity by different mechanisms, have been applied effectively in anti-cancer, anti-viral and anti-parasite therapies. A better understanding of the chemical mechanisms involved in normal catalysis and in inhibi- tion of the enzyme is important for the rational design of more specific and effective inhibitor compounds. To achieve this goal, computational methods, particularly quantum chemical calculations, have been used more and more frequently. The ever-growing capabilities of these methods together with undeniable advantages make it a stimulating area for research purposes. Introduction Ribonucleotide reductase (RNR) catalyzes the conver- sion of ribonucleotides into the correspondent 2¢-de- oxyribonucleotides, in the rate limiting step for the biosynthesis of DNA (Reichard 1993; Stubbe and van der Donk 1998). Ribonucleotide reductase is a ubiquitous radical- containing enzyme, which is usually classified into three different types, according to the cofactor needed to produce the essential organic radical, the active site residues that perform catalysis, and the phosphorylation state of the substrates. The Escherichia coli RNR is similar to the mammalian one (class I) and has served as its prototype in experimental and computational studies; these were much facilitated by the determination of the R1 and R2 subunits’ X-ray crystallographic structure (Eriksson et al. 1997; Nordlund et al. 1990; Uhlin and Eklund 1994). Escherichia coli RNR is an a 2 b 2 tetramer that results from the association of two catalytically inactive ho- modimers, R1 (a 2 ) and R2 (b 2 ) (see Fig. 1). Subunit R2 is the smaller of the two, with a molecular weight of 87 kDa, and contains a non-heme di-iron cluster, which is involved in generating and stabilizing the radical essential for catalysis. This radical is located in a tyro- sine residue (Tyr 122 ), it is formed upon dissociation of a molecule of O 2 by the binuclear ferrous [Fe(II)] complex, and is stabilized by the resulting oxo-bridged ferric [Fe(III)] dimer (see Fig. 2). The active site of the enzyme and the allosteric sites that regulate its specificity and activity are all located in the bigger R1 subunit, which has a molecular weight of 171 kDa. When the substrate (a ribonucleoside-5¢-diphosphate) binds to the active site, in R1, the radical character has to be transferred from the Tyr 122 in R2 to a cysteine residue (Cys 439 ) of the active site, which will in turn initiate the catalytic reaction. These two amino acid residues are ca. 35 A ˚ apart and the radical character relocation is believed to occur via hydrogen atom transfers along a pathway of conserved hydrogen-bonded residues. As for the catal- ysis itself, the active site residues directly involved in the set of reactions are three cysteines (Cys 439 , Cys 225 and Cys 462 ), one glutamate (Glu 441 ) (Persson et al. 1997) and one asparagine (Asn 437 ) (Kasrayan et al. 2002) (see Fig. 3). The reduction of the substrate leads to elimi- nation of a water molecule and oxidation of Cys 225 and Cys 462 to a disulfide bridge (Stubbe and van der Donk 1998). The same enzyme is responsible for the reduction of all four ribonucleotides. Its specificity towards each S. Pereira Æ N.M.F.S.A. Cerqueira Æ P. A. Fernandes M. J. Ramos (&) REQUIMTE/Departamento de Quı´mica, Faculdade de Cieˆncias do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal E-mail: pafernan@fc.up.pt Eur Biophys J (2006) 35: 125–135 DOI 10.1007/s00249-005-0026-6