2XO4

RIBONUCLEOTIDE REDUCTASE Y730NH2Y MODIFIED R1 SUBUNIT OF E. COLI

Summary for 2XO4

• Entry DOI: 10.2210/pdb2xo4/pdb
• Related: 1AV8 1BIQ 1JPR 1JQC 1MRR 1MXR 1PFR 1PIM 1PIU 1PIY 1PIZ 1PJ0 1PJ1 1PM2 1QFN 1R1R 1R65 1RIB 1RLR 1RNR 1RSR 1RSV 1XIK 1YFD 2ALX 2AV8 2R1R 2X0X 2XAK 2XAP 2XAV 2XAW 2XAX 2XAY 2XAZ 2XO5 3R1R 4R1R 5R1R 6R1R 7R1R
• Descriptor: RIBONUCLEOSIDE-DIPHOSPHATE REDUCTASE 1 SUBUNIT ALPHA, RIBONUCLEOSIDE-DIPHOSPHATE REDUCTASE 1 SUBUNIT BETA (3 entities in total)
• Functional Keywords: oxidoreductase, nucleotide-binding, alternative initiation, dna replication, allosteric enzyme
• Biological source: ESCHERICHIA COLI; More
• Total number of polymer chains: 7
• Total formula weight: 266761.87

Authors

Minnihan, E.C.,Seyedsayamdost, M.R.,Uhlin, U.,Stubbe, J. (deposition date: 2010-08-09, release date: 2010-08-18, Last modification date: 2023-12-20)

Primary citation

Minnihan, E.C.,Seyedsayamdost, M.R.,Uhlin, U.,Stubbe, J.
Kinetics of Radical Intermediate Formation and Deoxynucleotide Production in 3-Aminotyrosine- Substituted Escherichia Coli Ribonucleotide Reductases.
J.Am.Chem.Soc., 133:9430-, 2011

PubMed Abstract: Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y(122)•→ W(48)→ Y(356) in β2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of β. The resulting protein, Y(356)NH(2)Y-β2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y• formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y• formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

PubMed: 21612216
DOI: 10.1021/JA201640N

Experimental method

X-RAY DIFFRACTION (2.5 Å)