Yadav Lumbini R, Sharma Vasudha, Shanmugam Maheswaran, Mande Shekhar C
National Centre for Cell Science, SPPU Campus, Ganeshkhind, Pune, 411007, India.
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai, 400076, Maharashtra, India.
Curr Res Struct Biol. 2024 Sep 18;8:100157. doi: 10.1016/j.crstbi.2024.100157. eCollection 2024.
Class I ribonucleotide reductases consisting of α and β subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by di-manganese ions in the β subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the β subunits with no external support from another subunit. Class 1b ribonucleotide reductase complex is an essential enzyme complex in the human pathogen and its structural details are largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidised and reduced forms, and similarly those of NrdF2:NrdI complexes. These structures provide detailed atomic view of the mechanism of free radical generation in the β subunit in this pathogen. We observe a well-formed channel in NrdI from the surface leading to the buried FMN moiety and propose that oxygen molecule accesses FMN through it. The oxygen molecule is further converted to a superoxide ion upon electron transfer at the FMN moiety. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. The oxidation of Mn(II) in NrdF2I to high valent oxidation state (either Mn(III) or Mn(IV) assisted by the reduced FMN site was evidently confirmed by EPR studies. SEC-MALS and low resolution cryo-EM map indicate unusual stoichiometry of 2:1 in the NrdF2I complex. A density close to Tyr 110 at a distance <2.3 Å is observed, which we interpret as OH group. Overall, the study therefore provides important clues on the initiation of free radical generation in the β subunit of the ribonucleotide reductase complex in .
由α和β亚基组成的I类核糖核苷酸还原酶通过复杂的自由基机制将核糖核苷二磷酸转化为脱氧核糖核苷二磷酸。Ib类核糖核苷酸还原酶中自由基的产生由β亚基中的二价锰离子介导，并由类黄素氧还蛋白NrdI亚基提供外部辅助。这与Ia类核糖核苷酸还原酶不同，Ia类核糖核苷酸还原酶中自由基的产生始于β亚基中的双铁中心，没有来自另一个亚基的外部支持。1b类核糖核苷酸还原酶复合物是人类病原体中的一种必需酶复合物，其结构细节在很大程度上尚不清楚。在本研究中，我们确定了氧化态和还原态的分枝杆菌NrdI的晶体结构，以及NrdF2:NrdI复合物的晶体结构。这些结构提供了该病原体β亚基中自由基产生机制的详细原子视图。我们观察到NrdI中从表面通向埋藏的FMN部分的一个结构良好的通道，并提出氧分子通过它进入FMN。氧分子在FMN部分进行电子转移后进一步转化为超氧离子。同样，也观察到了超氧自由基在NrdI和NrdF2之间转移的途径。EPR研究明确证实，在还原的FMN位点辅助下，NrdF2I中的Mn(II)氧化为高价氧化态（Mn(III)或Mn(IV)）。SEC-MALS和低分辨率冷冻电镜图谱表明NrdF2I复合物中存在不寻常的2:1化学计量比。在距离<2.3 Å处观察到靠近Tyr 110的密度，我们将其解释为OH基团。因此，总体而言，该研究为该核糖核苷酸还原酶复合物β亚基中自由基产生的起始提供了重要线索。
