Ribonucleotide Class

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]. 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

The realization of the widespread occurrence of amino acid radicals in enzyme catalysis is recent and has been documented in several reviews (52-61). Among the catalytically essential redox-active amino acids glycyl [e.g., anaerobic class III ribonucleotide reductase (62) and pyruvate formate lyase (63-65)], tryptophanyl [e.g., cytochrome peroxidase (66-68)], cysteinyl [class I and II ribonucleotide reductase (60)], tyrosyl [e.g., class I ribonucleotide reductase (69-71), photosystem II (72, 73), prostaglandin H synthase (74-78)], and modified tyrosyl [e.g., cytochrome c oxidase (79, 80), galactose oxidase (81), glyoxal oxidase (82)] are the most prevalent. The redox potentials of these protein residues are well within the realm of those achievable by biological oxidants. These redox enzymes have emerged as a distinct class of proteins of considerable interest and research activity. 

As already mentioned, RNR is the metalloenzyme in which the first definitively characterized stable amino acid radical (1), later identified as a tyrosyl radical, was found in 1972. The RNR enzymes catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides utilized in DNA biosynthesis. There are three unique classes of this enzyme, differing in composition and cofactor requirements all of them, however, make use of metal ions and free radical chemistry. Excellent reviews on RNRs are available (60, 61, 70, 89-97). 

Figure 13.4 A proposed mechanism for all three classes of ribonucleotide reductases. Classes I and II RNRs require an active site Glu residue and a pair of redox-active Cys. Class HI RNRs lack the Glu and one of the Cys, and use formate as the reductant. (From Stubbe etal., 2001. Copyright 2001, with permission from Elsevier.)...

The two classes of nucleotide that must be synthesised are the pyrimidine and purine ribonucleotides for RNA synthesis and the deoxyribonucleotides for DNA synthesis. For the original sources of the nitrogen atoms in the bases of the pyrimidine and purine nucleotides, see Figure 20.7. The pathway for the synthesis of the pyrimidine nucleotides is... 

A new class of metalloprotelns containing polynuclear, non-heme oxo-bridged iron complexes has emerged recently. Dinuclear centers are present in hemerythrin (Hr), ribonucleotide reductase (RR), purple acid phosphatases (PAP) and, possibly, methane monooxygenase (MMO) these centers as well as model compounds are reviewed in Chapter 8. 

The enzymatic conversion of many analogues of the naturally occurring purines directly to their biologically active form, the ribonucleotides, in vivo [5, 8, 10, 13, 39] underlines the importance of these enzymes to the drug action of this class of compounds. 2-Aminoadenine (2, 6-diaminopurine, I) [107], 2-fluoroadenine (II) [108], 4-aminopyrazolo [3, 4-d] pyrimidine (VIll) [109]. and 2- and 8-aza-adenine (IX and X) [ 110, 111] have all been shown to be substrates for the adenine phosphoribosyltransferase [J12, 113]. Extensive studies on the metabolism of 2-aminoadenine (I) in E. coli [114, 115], L cells [116], and mice [117] have also shown its conversion by this enzyme to the ribonucleotide. 

A representative sampling of non-heme iron proteins is presented in Fig. 3. Evident from this atlas is the diversity of structural folds exhibited by non-heme iron proteins it may be safely concluded that there is no unique structural motif associated with non-heme iron proteins in general, or even for specific types of non-heme iron centers. Protein folds may be generally classified into several categories (i.e., all a, parallel a/)3, or antiparallel /8) on the basis of the types and interactions of secondary structures (a helix and sheet) present (Richardson, 1981). Non-heme iron proteins are found in all three classes (all a myohemerythrin, ribonucleotide reductase, and photosynthetic reaction center parallel a/)8 iron superoxide dismutase, lactoferrin, and aconitase antiparallel )3 protocatechuate dioxygenase, rubredoxins, and ferredoxins). This structural diversity is another reflection of the wide variety of functional roles exhibited by non-heme iron centers. 

RNR catalyzes the conversion of ribonucleotides to deoxyribonucleo-tides. Because of this central position in generating precursors for DNA synthesis, the enzyme has been the target for pharmacological modification. The reaction has been proposed to proceed through a radical mechanism involving the cyclic transfer of a preexisting radical in the enzyme to the substrate and back (Stubbe, 1989). Several classes of RNR have been identified on the basis of the type of radical and associated metallocenter. For the purposes of this chapter, only the E. coli enzyme, which has a tyrosyl radical and a binuclear iron cluster, is considered. 

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-... 

Dinuclear iron centres occur in several proteins. They either bind or activate dioxygen or they are hydrolases. Ribonucleotide reductase (RR) of the so-called class I type contains one such centre in the R2 protein in combination with a tyrosyl radical, both being essential for enzymatic activity which takes place in the R1 protein subunit. The diiron centre activates dioxygen to generate the tyrosyl radicals which in turn initiate the catalytic reaction in the R1 subunit. The interplay between the tyrosyl free radical in R2 and the formation of deoxyribonucleotides in R1 which also is proposed to involve a protein backbone radical is a topic of lively interest at present but is outside the scope of this review. Only a few recent references dealing with this aspect are mentioned without any further discussion.158 159 1 1,161... 

A second group of ribonucleotide reductases (Class II), found in many bacteria, depend upon the cobalt-containing vitamin B12 coenzyme which is discussed in Section B. These enzymes are monomeric or homodimeric proteins of about the size of the larger a subunits of the Class I enzymes. The radical generating center is the 5 -deoxyadenosyl coenzyme.350 364 365... 

Figure 16-21 (A) Scheme showing the diiron center of the R2 subunit of E. coli ribonucleotide reductase. Included are the side chains of tyrosine 122, which loses an electron to form a radical, and of histidine 118, aspartate 237, and tryptophan 48. These side chains provide a pathway for radical transfer to the R1 subunit where the chain continues to tyrosines 738 and 737 and cysteine 429.354a c From Andersson et al.35ic (B) Schematic drawing of the active site region of the E. coli class IH ribonucleotide reductase with a plausible position for a model-built substrate molecule. Redrawn from Lenz and Giese373 with permission.

RNA consists of long strings of ribonucleotides, polymerised in a similar way to DNA, but the chains are considerably shorter than those of DNA. RNA contains ribose rather than deoxyribose and also contains uracil instead of thymidine. This has important connotations in the secondary structure of RNA which does not form the long helices found in DNA. RNA is usually much more abundant than DNA in the cell and its concentration varies according to cell activity and growth. This is because RNA has several roles in protein synthesis. There are three major classes messenger RNA (mRNA) ribosomal RNA (rRNA) and transfer RNA (tRNA). 

The reactions catalyzed by B12 may be grouped into two classes those catalyzed by methylcobalamin and those catalyzed by cofactor B,2. The former reactions include formation of methionine from homocysteine, methanogenesis (formation of methylmercury is an important side reaction), and synthesis of acetate from carbon dioxide (82). The latter reactions include the ribonucleotide reductase reaction and a variety of isomerization reactions (82). Since dehydration and deamination have been studied quite extensively and very possibly proceed via [Pg.257]

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)...

Stubbe J, Nocera DG, Yee CS, Chang MCY. Radical initiation in the class I ribonucleotide reductase long-range proton-coupled electron transfer Chem Rev 2003 103 2167-201. 

Cho KB, V Pelmenschikov, A Graslund, PEM Siegbahn (2004) Density functional calculations on class III ribonucleotide reductase Substrate reaction mechanism with two formates. J. Phys. Chem. B 108 (6) 2056-2065... 

Ribonucleotide Reductase. All living organisms depend upon one of three classes of ribonucleotide reductases in the biosynthesis of... 

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