PDF(Pubmed)
Abstract：
A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
摘要：
一个小,核苷酸结合域，ATP锥，在大多数核糖核苷酸还原酶（RNR）催化亚基的N末端发现。通过结合三磷酸腺苷(ATP)或三磷酸脱氧腺苷(dATP),其调节所有类型的RNR的酶活性。对需氧RNR的功能和结构工作揭示了多种方式，其中dATP通过诱导寡聚化并阻止从一个亚基到另一个亚基的活性位点的生产性自由基转移来抑制活性。无氧RNRs,另一方面,在活性位点旁边储存稳定的甘氨酰自由基，其dATP依赖性抑制的基础是完全未知的。我们展示了生化，生物物理，以及有关ATP和dATP与Prevotellacopri厌氧RNR结合作用的结构信息。当两个ATP分子与ATP-锥结合时，酶以二聚体-四聚体平衡存在，而当两个dATP分子结合时，酶则偏向二聚体。在ATP存在的情况下,P.copriNrdD是有活性的并且在二聚体的一个单体中具有完全有序的甘氨酰自由基结构域(GRD)。dATP与ATP-cone的结合导致GRD的活性丧失和动力学增加。使得它不能在低温EM结构中被检测到。甘氨酰自由基甚至以dATP结合的形式形成，但底物不结合。这些结构暗示了活性调节中相互作用的复杂网络，该网络涉及GRD距dATP分子30多埃，变构底物特异性位点和活性位点上保守但以前看不见的瓣。一起来看,结果表明，dATP在厌氧RNR中的抑制作用通过增加皮瓣和GRD的柔韧性而起作用，从而防止底物结合和自由基动员。
