Ribonucleotide reductase of E. coli

Ribonucleotide reductase of E. coli consists of two non-identical subunits, which are purified together during the initial steps of the purification procedure however upon further purification the reductase separates into two subunits, proteins B1 and B2 (58). The overall yield of the two proteins is low, in particular that of protein Bl, which readily decomposes into smaller subunits. This decomposition can be counteracted by the addition of dithiols. An improved procedure for the purification of protein B1 using affinity chromatography on dATP-Sepharose has been described by Thelander (59). 

All non-heme iron containing ribonucleotide reductases are also inhibited by hydroxyurea and related hydroxamates, while the adenosyl-cobalamin-dependent reductases are not affected (27, 156). The inhibition by these reagents can be partially reversed by excess Fe+2 or dithiols. Reaction of ribonucleotide reductase of E. coli with [14C]hydro-xyurea inactivated only the B2 subunit and this inactivation was not reversed by removal of the radioactivity (157). Inactivation by hydroxyurea does not affect the iron content of protein B2, but involves the destruction of the stable free radical (66,67). Reactivation can be accomplished by removal of the iron and reconstitution of apoprotein B2 with Fe+2. Hydroxyurea has been demonstrated to be a powerful radical scavenger in another system (158). 

The ribonucleotide reductase of E. coli is an allosteric enzyme, the activity and specificity of which are modulated in a very complex manner by several nucleoside triphosphates. Both the Michaelis constant and Fmax values for the individual substrates were found to be influenced by such... 

Figure 7. ESR spectra ( —196°C) of tyrosyl radicals in ribonucleotide reductase from E. coli. a, Spectrum from enzyme grown in the presence of tyrosine b, spectrum from enzyme grown in the presence of deuterated [yJ./i- HjJtyrosine. From [137], with permission.

Thioredoxin from yeast has been obtained in two forms (I and II) of which thioredoxin II has been purified to homogeneity (43). Both yeast thioredoxins are able to serve as hydrogen donors for the ribonucleotide reductase from E. coli. The molecular weight (12,600) of thioredoxin II is similar to that of thioredoxin from E. coli. Although both yeast thioredoxins contain only one tryptophan residue and although their amino acid compositions differ markedly from that of E. coli thioredoxin, the amino acid sequences around the disulfide bridge of these three thioredoxins are identical ... 

During purification, the ribonucleotide reductase from E. coli separates into two fractions, B1 and B2. These fractions have been purified to virtual homogeniety separately they are catalytically inactive, but together, in the presence of Mg +, a catalytically active complex is formed consisting... 

Fig, 16-1. Influence of allosteric effectors on reduction of GDP by ribonucleotide reductase from E. coli. From (6). Reproduced with permission. 

In a PCET, the electron does not have to be coupled to a particular proton throughout the entire reaction.A prominent example is the class I ribonucleotide reductase from E. coli, which catalyses the reduction of nucleoside diphosphates (NDPs) to deoxynucleotide diphosphates (dNDPs), the building blocks of deoxyribonucleic acid (DNA). An electron is transferred from a... 

A four-pulse DEER measurement of the distance between two tyrosyl radicals on the monomers that make up the R2 subunit of E. coli ribonucleotide reductase gave a point-dipole distance of 33.1 A, which is in good agreement with the X-ray crystal structure.84 Better agreement between the calculated and observed dipolar frequency could be obtained by summing contributions from distributed... 

Eliasson, R., Pontis, E., Sun, X., Reichard, P. (1994) Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli. J. Biol. Chem. 269, 26,052-26,057. 

The resulting thiol pair of the reduced thioredoxin is the reductant used for ribonucleotide reductase (Chapter 16). The standard redox potential E° of E. coli thioredoxin is -0.27 V, appropriately low for coupling to the NADPH / NADP+ system. 

It was a surprise to discover that a mutant of E. coli lacking thioredoxin can still reduce ribonucleotides. In the mutant cells thioredoxin is replaced by glutaredoxin, whose active site disulfide linkage is reduced by glutathione rather than directly by NADPH. Oxidized glutathione is, in turn, reduced by NADPH and glutathione reductase. Thus, the end result is the same with respect to ribonucleotide reduction. 

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.

Ribonucleotide reductases are discussed in Chapter 16. Some are iron-tyrosinate enzymes while others depend upon vitamin B12, and reduction is at the nucleoside triphosphate level. Mammalian ribonucleotide reductase, which may be similar to that of E. coli, is regarded as an appropriate target for anticancer drugs. The enzyme is regulated by a complex set of feedback mechanisms, which apparently ensure that DNA precursors are synthesized only in amounts needed for DNA synthesis.273 Because an excess of one deoxyribonucleotide can inhibit reduction of all... 

Three types of ribonucleotide reductase catalyze this reduction of the ribose ring. The most widely distributed in nature occurs in mammalian and plant cells, in yeast, and in some prokaryotes. This type of reductase contains a tyrosyl radical closely associated with nonheme iron, as in the reductase from E. coli. The E. coli reductase is composed of two nonidentical subunits, both contributing to the active site it is specific for the reduction of diphosphates (ADP, GDP, CDP, and UDP). 

Figure 1. Structure of the R2 protein of E. coli ribonucleotide reductase in the met form. (Adapted from reference 7, which reports the 2.2 A crystal structure of the protein. Note that, in reference 7, Asp 84 is considered to be bidentate and chelating, but we prefer the mono dentate, hydrogen-bonded representation depicted above based on an analysis of the Fe-O distances.)...

Figure 2. Structure of a S211A mutant of the R2 protein of E. coli ribonucleotide reductase in the reduced form. (Adapted from reference 11 reporting the 2.2 A crystal structure.)...

Bollinger, J. M., Tong, W. H., Ravi, N., Huynh, B. H., Edmondson, D. E., and Stuhhe, J., 1994h, Mechanism of assembly of the tyrosyl radical-diiron(III) cofactor of E. coli ribonucleotide reductase III. Kinetics of the limiting Fe reaction by optical, EPR, and M ssbauer spectroscopies. /. Am. Chem. Soc. 116 8024n8032. 

Silva, K. E., Elgren, T. E., Que, L., and Stankovich, M. T., 1995, Electron transfer properties of the R2 protein of ribonucleotide reductase from Escherichia coli. Biochemistry 34 14093nl4103. 

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