Purification of ribonucleotide reductase subunits Y1, Y2, Y3, and Y4 from yeast: Y4 plays a key role in diiron cluster assembly.
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two types of subunits: RNR1 contains the active site for reduction and the binding sites for the nucleotide allosteric effectors. RNR2 contains the diiron-tyrosyl radical (Y.) cofactor essential for the reduction process. Studies in yeast have recently identified four RNR subunits: Y1 and Y3, Y2 and Y4. These proteins have been expressed in Saccharomyces cerevisiae and in Escherichia coli and purified to approximately 90% homogeneity. The specific activity of Y1 isolated from yeast and E. coli is 0.03 micromol.min(-1).mg(-1) and of (His)(6)-Y2 [(His)(6)-Y2-K387N] from yeast is 0.037 micromol. min(-1).mg(-1) (0.125 micromol.min(-1).mg(-1)). Y2, Y3, and Y4 isolated from E. coli have no measurable activity. Efforts to generate Y. in Y2 or Y4 using Fe(2+), O(2), and reductant have been unsuccessful. However, preliminary studies show that incubation of Y4 and Fe(2+) with inactive E. coli Y2 followed by addition of O(2) generates Y2 with a specific activity of 0.069 micromol.min(-1). mg(-1) and a Y. A similar experiment with (His)(6)-Y2-K387N, Y4, O(2), and Fe(2+) results in an increase in its specific activity to 0.30 micromol.min(-1).mg(-1). Studies with antibodies to Y4 and Y2 reveal that they can form a complex in vivo. Y4 appears to play an important role in diiron-Y. assembly of Y2.
Nguyen
H H
HH
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Ge
J
J
Perlstein
D L
DL
Stubbe
J
J
D001483:Base Sequence; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D007501:Iron / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D017931:DNA Primers; D011994:Recombinant Proteins; D007501:Iron; D012264:Ribonucleotide Reductases