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BIOLOGICAL SCIENCES / BIOCHEMISTRY
Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, M407 Walters Life Sciences, Knoxville, TN 37996-0840
Communicated by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, January 17, 2006 (received for review October 10, 2005)
Ribonucleotide reductase catalyzes a crucial step in de novo DNA synthesis and is allosterically controlled by relative levels of dNTPs to maintain a balanced pool of deoxynucleoside triphosphates in the cell. In eukaryotes, the enzyme comprises a heterooligomer of 
2 and 
2 subunits. The subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic subunit of ribonucleotide reductase from Saccharomyces cerevisiae. The structures of the apo-, AMPPNP only-, AMPPNP–CDP-, AMPPNP–UDP-, dGTP–ADP- and TTP–GDP-bound complexes give insight into substrate and effector binding and specificity cross-talk. These are Class I structures with the only fully ordered catalytic sites, including loop 2, a stretch of polypeptide that spans specificity and catalytic sites, conferring specificity. Binding of specificity effector rearranges loop 2; in our structures, this rearrangement moves P294, a residue unique to eukaryotes, out of the catalytic site, accommodating substrate binding. Substrate binding further rearranges loop 2. Cross-talk, by which effector binding regulates substrate preference, occurs largely through R293 and Q288 of loop 2, which are analogous to residues in Thermotoga maritima that mediate cross-talk. However loop-2 conformations and residue–substrate interactions differ substantially between yeast and T. maritima. In most effector–substrate complexes, water molecules help mediate substrate–loop 2 interactions. Finally, the substrate ribose binds with its 3' hydroxyl closer than its 2' hydroxyl to C218 of the catalytic redox pair. We also see a conserved water molecule at the catalytic site in all our structures, near the ribose 2' hydroxyl.
ribonucleotide reductase | allosteric regulation | crystallography | DNA synthesis | dNTP pools
*Present address: Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115-5730.
Author contributions: C.D. designed research; H.X., C.F., T.U., J.W.F., J.R., and C.D. performed research; H.X., C.F., T.U., J.R., and C.D. contributed new reagents/analytic tools; H.X., C.F., T.U., J.W.F., and C.D. analyzed data; and H.X., C.F., and C.D. wrote the paper.
Conflict of interest statement: No conflicts declared.
Data deposition: The atomic coordinates reported in this paper have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 1ZYZ (apo), 2CVS (native), 2CVT (complex with AMPPNP), 2CVU (complex with AMPPNP and CDP), 2CVV (complex with AMPPNP and UDP), 2CVW (complex with TTP and GDP), and 2CVX (complex with dGTP and ADP)].
To whom correspondence should be addressed. E-mail: cdealwis{at}utk.edu
© 2006 by The National Academy of Sciences of the USA
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