Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (Mn(IV)/Fe(IV)) Chlamydia trachomatis ribonucleotide reductase.
Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (Mn(IV)/Fe(IV)) Chlamydia trachomatis ribonucleotide reductase.
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DOI：
10.1021/ja309468s
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发表时间：
2012-12-19
影响因子：
15
通讯作者：
Krebs, Carsten
中科院分区：
文献类型：
--
作者：
Dassama, Laura M. K.;Jiang, Wei;Varano, Paul T.;Pandelia, Maria-Eirini;Conner, Denise A.;Xie, Jiajia;Bollinger, J. Martin, Jr.;Krebs, Carsten
I类核糖核苷酸还原酶（RNR）利用β亚基中的酪氨酰自由基（Y·）或MnIV/FeIII簇氧化α亚基中的半胱氨酸残基，产生从底物中夺取氢（H·）的硫酰基自由基。对于任一种氧化剂，亚基间“空穴转移”或“自由基易位”（RT）过程被认为是通过“跳跃”机制发生的，该机制涉及沿着特定途径的多个酪氨酰基（可能还有一个酪氨酰基）自由基中间体沿着。跳跃中间体从来没有直接检测到Mn/Fe依赖（Ic类）RNR，也没有在任何野生型RNR。沙眼衣原体RNR的MnIV/FeIII辅因子通过MnIV/FeIV中间体组装。在这里，我们表明，当α存在时，这种辅因子组装中间体可以将空穴传播到RT通路中，积累具有Y·s特征的EPR谱的自由基。Y·积累对底物存在的依赖性表明，这种“超氧化”酶形式中的RT是由蛋白质门控的，并且具有被苯丙氨酸取代的亚基界面途径Y的β变体不能支持自由基积累意味着野生型酶中的Y·（s）存在于RT途径中。值得注意的是，两种具有使其在MnIV/FeIII状态下失活的途径取代的变体β蛋白可以在其MnIV/FeIV状态下产生途径Y·s，并且还影响核苷酸还原。因此，使用更多的氧化辅因子允许跳跃中间体的积累和RT途径中的工程缺陷的“跨越”。
A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y•) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼ 35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H•) from the substrate. With either oxidant, the inter-subunit “hole transfer” or “radical translocation” (RT) process is thought to occur by a “hopping” mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y•s. The dependence of Y• accumulation on the presence of substrate suggests that RT within this “super-oxidized” enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y•(s) in the wild-type enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y•s in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the “hurdling” of engineered defects in the RT pathway.
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影响因子：
15
作者：
BENDER, CJ;SAHLIN, M;SJOBERG, BM
通讯作者：
SJOBERG, BM
影响因子：
56.9
作者：
Högbom, M;Stenmark, P;Nordlund, P
通讯作者：
Nordlund, P
影响因子：
2.9
作者：
Jiang, Wei;Hoffart, Lee M.;Martin Bollinger, J., Jr.
通讯作者：
Martin Bollinger, J., Jr.
影响因子：
56.9
作者：
Jiang, Wei;Yun, Danny;Bollinger, J. Martin, Jr.
通讯作者：
Bollinger, J. Martin, Jr.
影响因子：
5.6
作者：
BROWN, NC;REICHARD, P
通讯作者：
REICHARD, P