Ribonucleotide reductase purification

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. 

Mann, G. J., Gr%oslund, A., Ochiai, E., Ingemarson, R., and Thelander, L., 1991, Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry 30 1939nl947. 

Nguyen, H. H., Ge, J., Perlstein, D. L., and Stubbe, J., 1999, Purification of ribonucleotide reductase subunits Yl, Y2, Y3, and Y4 from yeast Y4 plays a key role in diiron cluster assembly. Proc. Natl. Acad. Sci. USA 96 12339iil2344. 

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). 

Additional support for the Mn specificity of native B. ammoniagenes ribonucleotide reductase is provided by its inhibition by cyanide (Fig. 5). The iron- and B 12-enzymes are not affected by cyanide which forms cyano and cyanoaquo complexes with Mn" but not with Fe or Fe" " " under typical enzyme assay conditions at ambient temperature. We thus believe that manganese in the Brevibacterium enzyme system is the functional, catalytic counterpart of iron found in E. coli and eukaryotic ribonucleotide reductases. The nature of Mn -protein interaction remains to be studied after further purification. 

Whether or not pyrimidine and purine ribonucleotides are reduced by a single enzyme is a matter of some controversy. The monomeric reductase of L. leichmannii and the enzyme of E. coll where substrates bind exclusively to Bas well as calf thymus and human reductase consist of only one holoenzyme species capable to reduce all the ribonucleotides. In contrast rat liver ribonucleotide reductase during purification dissociated into one protein fraction specific for CDP reduction and one reducing the three other diphosphates from which a specific UDP reductase activity could then be obtained Other reports of differential ADP and CDP reductase activities in rodent enzyme preparations are difficult to assess. In reductase assays one can easily encounter slow reduction rates for one particular ribonucleotide when not all conditions like pH, ionic strength, effector nucleotide and dithiol concentrations, and stoichiometry of subunits, are at an optimum. Therefore differential activities not necessarily indicate different enzyme species. 

As purification of the ribonucleotide reductase progressed, it became evident that the activity was present in a complex of two protein components, B1 and B2 these are now known to be nonidentical subunits of a remarkable allosteric enzyme. It was recognized that dihydrolipoate was unlikely to be the physiological reductant in this system. 

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... 

Purification of the steroid-active isoenzyme of alcohol dehydrogenase by affinity chromatography Isolation of ribonucleotide reductase by affinity chromatography Investigations of the effects of different spacer arms on the affinity chromatography of alkaline phosphatase Purification of P-450 haemeproteins of Rhizobium japonicum by affinity chromatography... 

Ribonucleotide reductase, an allosteric enzyme from Escherichia coli, converts ribonucleotide diphosphates to the corresponding deoxyribo-nucleotides, and therefore provides the necessary precursors for DNA synthesis (Reichard, 1967). During purification ribonucleotide reductase separates into two nonidentical subunits, proteins B1 and B2, each enzymatically inactive (Brown et a/., 1969a). The active enzyme is formed in the presence of magnesium ions and consists of a 1 1 complex of the two subunits (Thelander, 1973). Proton subunit B2 (mol. wt. 78,000), which participates in the formation of the catalytic site (Thelander et al, 1976), contains nonheme iron (Brown et al, 1969b) and an ESR-detectable organic... 

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