Ribonucleotide reductase RNR

Rubrerythrin (Rr) was first isolated in 1988 from cellular extracts of D. vulgaris Hildenborough (38), and later also found in D. desulfuri-cans (39). Rr is constituted by two identical subunits of 22 kDa and it was shown that each monomer contains one Rd-like center, Fe(RS)4, and a diiron-oxo center similar to the ones found in methane monooxygenase (MMO) (40, 41) or ribonucleotide reductase (RNR-R2) (42). After aerobic purification, the UV-visible spectrum shows maxima at 492, 365, and 280 nm, and shoulders at 570 and 350 nm. This spectrum is similar to the ones observed for Rd proteins. From a simple subtraction of a typical Rd UV-vis spectrum (normalized to 492 nm) it is possible to show that the remainder of the spectrum (maxima at 365 nm and a shoulder at 460 nm) strongly resembles the spectrum of met-hemerythrin, another diiron-oxo containing protein. 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

Metalloenzymes with non-heme di-iron centers in which the two irons are bridged by an oxide (or a hydroxide) and carboxylate ligands (glutamate or aspartate) constitute an important class of enzymes. Two of these enzymes, methane monooxygenase (MMO) and ribonucleotide reductase (RNR) have very similar di-iron active sites, located in the subunits MMOH and R2 respectively. Despite their structural similarity, these metal centers catalyze very different chemical reactions. We have studied the enzymatic mechanisms of these enzymes to understand what determines their catalytic activity [24, 25, 39-41]. 

Domino reactions are not only useful for the construction of molecules, but also for their degradation. This concept is often encountered in nature. Thus, ribonucleotide reductases (RNRs) are enzymes that catalyze the formation of DNA monomers from ribonucleotides by radical mediated 2 -deoxygenation. This process has also been studied... 

Figure 13.2 The reaction catalysed by ribonucleotide reductase (RNR). (From Stubbe et al., 2001. Copyright 2001, with permission from Elsevier.)...

Initial insight of the role of CSN in cell-cycle control came from the finding that csnl and csn2 deletion S. pomhe strains have an S-phase delay [52]. Interestingly, this effect did not occur in strains missing other CSN subunits. The S-phase delay was caused by the accumulation of the cell-cycle inhibitor Spdl (S-phase delayed 1), which is involved in the misregulation of the ribonucleotide reductase (RNR). RNR catalyzes the production of deoxyribonucleotides for DNA synthesis and... 

Proteins with dinuclear iron centres comprise some well studied representatives like ribonucleotide reductase (RNR), purple acid phosphatase (PAP), methane monooxygenase hydroxylase (MMOH), ruberythrin and hemerythrin. The latter is an oxygen carrier in some sea worms it has been first characterized within this group and has thus laid the foundation to this class of iron coordination motif. Ruberythrin is found in anaerobic sulfate-reducing bacteria. Its name implies that, in addition to a hemerythrin-related diiron site another iron is coordinated in a mononuclear fashion relating to rubredoxin which is an iron-... 

Figure 6 High Field EPR spectra of radicals occurring in PS II ( Yz obtained at 245 GHz, all other spectra at 285 GHz). For comparison, the spectra of the tyrosine radical in ribonucleotide reductase (RNR) and of irradiated tyrosine hydrochloride crystals n are also shown (for details see reference 30). Note the striking differences in g values for the tyrosyl radicals. Figure reproduced from reference 30 with permission.

Scheme 2.6 Examples of reactions catalyzed byenzymesthat carry a dinudear iron active site (a) hydroxylation of methane by soluble methane monooxygenase (sMMO) [7] (b) reduction of ribonucleotides by class I ribonucleotide reductase (RNR)...

Figure 6 Reactions catalyzed by ribonucleotide reductase (RNR). HS-E-SH is the site of the enzyme responsible for the reduction step SH is a cysteine thiol group.

Siegbahn PEM, Eriksson L, Himo F, Pavlov M. Hydrogen Atom Transfer in Ribonucleotide Reductase (RNR). J Phys Chem B. 1998 102 10622-9. 

Figure 1. Comparison of the coordination spheres of the dinuclear iron sites in oxidized hemerythrin (Hr), ribonucleotide reductase (RNR), uteroferrin (Uf), and methane monooxygenase (MMO).

Figure 7. Proposed proton-coupled electron transfer (PCET) pathway between the R2 and R1 subunits of the E. coli ribonucleotide reductase (RNR) complex. The conserved amino acids are shown schematically E. coli numbering).

Ribonucleotide reductases (RNR) constitute a large group of essential enzymes with a diverse array of primary as well as quaternary structures. Common for the enzymes is that they catalyze the rate-determining step in DNA biosynthesis, the reduction of ribonucleotides into deoxy-ribonucleotides (Figure 19) [44,45]. 

An unprecedented example of the application of an organic azide as an enzyme inhibitor derives from the elegant studies of Stubbe and coworkers at MIT, who have investigated the mechanism of action of ribonucleotide reductase (RNR) using several mechanism-based inhibitors including 2 -azido-2 -deoxyuridine-5 -diphosphate (NjUDP) (71) [82]. RNR plays a... 

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