Ribonucleotides reduction

From these data it seems feasible that a Co(II)-species is generated during catalysis, and that homolysis of the Co—C-bond is a prerequisite for enzyme catalysis in ribonucleotide reductase. However, the kinetics of appearance of the Co(II)-signal indicates that the rate of formation of Co(II) is much slower than either the rate of ribonucleotide reduction... 

Biosynthetic production of thymidine is overall a complex process combining the controlled introduction of a novel biotransformation step into a biological system with selective enhancement or knock-out of a series of existing metabolic steps. Metabolic engineering to enhance cofactor recycling at both ribonucleotide reduction and dUMP methylation steps has important parallels in other systems, as whole-cell biotransformations are frequently employed as a means to supply, in situ, high-cost and usually labile cofactors. 

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. 

The tyrosyl radical is used to initiate the ribonucleotide reduction at the active site in the R1 protein 3.5 nm away. The tyrosyl radical is very stable and was discovered by a characteristic EPR spectrum of isolated enzyme. Alteration of this spectrum when bacteria were grown in deuterated tyrosine indicated that the radical is located on a tyrosyl side chain and that the spin density is delocalized over the tyrosyl ring.361 362 Using protein engineering techniques the ring was located as Tyr 122 of the E. coli enzyme. A few of the resonance structures that can be used to depict the radical are the following ... 

Hogenkamp, H. P. C., Ghambeer, R. K., Brownson, C., Blakley, R. L., and Vitols, E., 1968, Cobamides and ribonucleotide reduction. VI. Enzyme-catalyzed hydrogen exchange between water and deoxyadenosylcobalamin. J. Biol. Chem. 243 7999808. 

Tamao, Y., and Blakely, R. L., 1973, Direct spectroscopic observation of an intermediate formed from deoxyadenosylcobalamin in ribonucleotide reduction. Biochemistry 12 24034. 

Gerfen, G. J., Licht, S., Willems, J. P., Hoffman, B. M., and Stubbe, J., 1996, Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction Evidence for a thiyl radical-Cob(II) alamin interaction. J. Am. Chem. Soc. 118 819298197. 

Reichard, P., 1997, The evolution of ribonucleotide reduction. Trends. Biochem. Sci. 22 81n85. 

The active site similarities listed above belie a remarkable functional diversity, which includes phosphate ester hydrolysis, dioxygen and NO reduction, reversible O2 binding, and O2 activation, the last of which includes enzymes involved in ribonucleotide reduction, hydrocarbon monooxygenation, and fatty acyl desaturation. At the overall protein level, the purple acid phosphatases (PAPs) seem to be completely unrelated, both structurally and functionally, to any of the others in this class. Similarly, the flavo-diiron enzymes form a structurally and probably functionally distinct family of proteins, catalyzing both dioxygen and NO reduction. These last two examples illustrate that attempts to shoehorn all of these enzymes into a single class can sometimes provide a simplistic and misleading view of their chemistry and biochemistry. 

The observation that these deoxyribonucleotide precursors exist in very low concentration in cells suggests that ribonucleotide reduction is a crucial and rate-controlling step in DNA synthesis and cell division. Indeed the level of reductase activity has been correlated with DNA replication (7), cell proliferation (2) and tumor growth rate (3, 4). 

The reduction of ribonucleotides to deoxyribonucleotides is linked to NADPH by thioredoxin and thioredoxin reductase. Thioredoxin is a small protein which is oxidized from a dithiol to a disulfide form during ribonucleotide reduction. The dithiol form of thioredoxin is regenerated by NADPH and a specific flavoprotein, thioredoxin reductase. The thioredoxin system consisting of thioredoxin and thioredoxin reductase was first identified as the reducing system from E. coli by Reichard and co-workers (35, 36) and both proteins have since been purified to homogeneity (37, 38). 

Very recently ribonucleotide reduction has been reviewed by Henderson and Paterson (46) and by Follmann (47) earlier reviews are those by Reichard and co-workers (48—51) and by Blakley (52). Ribonucleotide reduction has also been covered in several issues of Annual Review of Biochemistry (53—56). 

The effect of ara-ATP and ara-CTP on ribonucleotide reduction has also been studied in crude extracts from Novikoff (139) and Ehrlich (140) ascites tumor cells. Inhibition by these nucleotide analogs appeared to be competitive with effector nucleotides, but not with nucleoside diphosphate substrates. 

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