Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

Abstract

The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y • ) cofactor. In Saccharomyces cerevisiae, two R1s, α n and , have been identified, while R2 is a heterodimer (ββ′). β′ cannot bind iron and generate the Y • ; consequently, the maximum amount of Y • per ββ′ is 1. To determine the cofactor stoichiometry in vivo, a FLAGtagged β ( FLAG β) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of FLAG ββ′ by immunoaffinity chromatography, which was found to have 0.45 ± 0.08 Y • / FLAG ββ′ and a specific activity of 2.3 ± 0.5 μmol min −1 mg −1 . FLAG ββ′ isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y • / FLAG ββ′ ratio of 0.5. To determine the Y • /ββ′ ratio without R2 isolation, whole cell EPR and quantitative Western blots of β were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y • /ββ′ ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Δ gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y • /ββ′ ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y • and modulation of Y • concentration is not involved in the regulation of RNR activity.

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

“…Interestingly, purified recombinant Spd1 protein has been shown to bind the S. pombe R1 subunit and inhibits its activity in vitro, while Spd1-R2 interaction cannot be detected under the same conditions (16). However, the measured specific activity of purified S. pombe R1 in the report (ϳ10 nmol dCDP/mg/min [16]) is extremely low relative to that of the S. cerevisiae R1 (250 nmol dCDP/mg/min [16]; ϳ800 nmol dCDP/mg/min [35]) and the mouse R1 (130 nmol dCDP/mg/min [16]), suggesting low levels of active S. pombe R1 proteins in the preparation. Hence, the mechanistic basis for Spd1-mediated RNR inhibition remains to be elucidated.…”

“…Only Rnr2 is capable of forming the (Fe) 2 -Y ⅐ cofactor (36). Rnr4 is incapable of forming any radical, as it contains substitutions in three of the six conserved residues required for iron binding (17,35,48). Nevertheless, Rnr4 is required to facilitate the generation of radicals in Rnr2 and stabilizes the resulting heterodimer both in vitro and in vivo (7,36,46).…”

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

“…The basis for the activity differences are not understood, but are likely related to the complexity of the assay as we articulated in detail in ref. 32. CDP, 0.19 and 0.08 eq were detected in the absence or presence of denaturant, respectively.…”

“…The instability of the ␤2 radical requires that a control in the absence of F 2 CDP be carried out and used to correct the data observed in its presence. The weak interaction between ␣ and ␤ of RNR allow assays of the subunit activity individually (21,32). Thus, the inactivation mixture was assayed for activity of ␣ (␤) in the presence of a 7-fold excess of ␤ (␣).…”

Investigation of in Vivo Diferric Tyrosyl Radical Formation in Saccharomyces cerevisiae Rnr2 Protein

“…Ϫ1 when assayed in the presence of a 10ϫ molar excess of FLAG-␤␤Ј (specific activity ϳ3000 nmol min Ϫ1 mg Ϫ1 ) from yeast ⌬crt1 MHY619 cells (43). Untagged ␤Ј, N-terminally His 6 -tagged ␤Ј, and N-terminally His 6 -tagged ␤Ј with the C-terminal 8 amino acids removed (His 6 -␤Ј⌬8aa) were purified using anion exchange column and nickel-nitrilotriacetic acid column, respectively (18,42).…”