Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Minnihan E.C.; Ando N.; Brignole E.J.Olshansky L.Chittuluru J.Asturias F.J.Drennan C.L.Nocera D.G.Stubbe J.

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Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

机译：Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

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Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y?) in the RNR small (β2) subunit over a 35-? pathway of redox-active amino acids: Y_(122)? ? W_(48)? ? Y_(356) in β2 to Y_(731) ? Y_(730) ? C_(439) in α2. When 3-aminotyrosine (NH_2Y) is incorporated in place of Y_(730), a long-lived NH_2Y_(730)? is generated in α2 in the presence of wildtype (wt)-β2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH_2Y_(730)? induces formation of a kinetically stable α2β2 complex. Under conditions that generate NH2Y730?, binding between Y730NH2Y-α2 and wt-β2 is 25-fold tighter (K_d = 7 nM) than for wt-α2wt-β2 and is cooperative. Stopped-flow fluorescence experiments establish that the dissociation rate constant for the Y_(730)NH_2Y-α2wt- β2 interaction is ～10~4-fold slower than for the wt subunits (～60 s-1). EM and small-angle X-ray scattering studies indicate that the stabilized species is a compact globular α2β2, consistent with the structure predicted by Uhlin and Eklund's docking model Uhlin U, Eklund H (1994) Nature 370(6490):533-539. These results present a structural and biochemical characterization of the active RNR complex "trapped" during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2013年第10期|3835-3840|共6页
作者
Minnihan E.C.; Ando N.; Brignole E.J.Olshansky L.Chittuluru J.Asturias F.J.Drennan C.L.Nocera D.G.Stubbe J.;
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作者单位

Departments of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States;

Scripps Research Institute, Department of Cell Biology, San Diego, CA 92037, United States;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
Conformational equilibria; Radical transfer; Unnatural amino acid;

Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

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