PDF(Pubmed)
Abstract：
The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non-trivial topology, namely, a 31 knot in the active site. Here, we address the issue of seemingly unfeasible deprotonation of Ψ in Nep1 active site by a distant aspartate residue (D101 in S. cerevisiae), using a combination of bioinformatics, computational, and experimental methods. We identified a conserved hydroxyl-containing amino acid (S233 in S. cerevisiae, T198 in A. fulgidus) that may act as a proton-transfer mediator. Molecular dynamics simulations, based on the crystal structure of S. cerevisiae, and on a complex generated by molecular docking in A. fulgidus, confirmed that this amino acid can shuttle protons, however, a water molecule in the active site may also serve this role. Quantum-chemical calculations based on density functional theory and the cluster approach showed that the water-mediated pathway is the most favorable for catalysis. Experimental kinetic and mutational studies reinforce the requirement for the aspartate D101, but not S233. These findings provide insight into the catalytic mechanisms underlying proton transfer over extended distances and comprehensively elucidate the mode of action of Nep1.
摘要：
Nep1蛋白对于真核和古细菌小核糖体亚基的形成至关重要，并且它在pre-rRNA加工过程中催化假尿苷的位点定向SAM依赖性甲基化（Φ）。它具有非平凡的拓扑结构，即,活动部位有31个结。这里,我们解决了似乎不可行的去质子化的问题，在Nep1活性位点通过一个遥远的天冬氨酸残基（D101在酿酒酵母），结合生物信息学，计算，和实验方法。我们确定了一种保守的含羟基氨基酸(酿酒酵母中的S233，T198在A.fulgidus中），可以充当质子转移介质。分子动力学模拟,根据酿酒酵母的晶体结构，以及在A.fulgidus中通过分子对接产生的复合物上，证实这种氨基酸可以穿梭质子，然而,活性位点中的水分子也可以起到这种作用。基于密度泛函理论和簇方法的量子化学计算表明，水介导的途径最有利于催化。实验动力学和突变研究加强了对天冬氨酸D101而不是S233的需求。这些发现提供了对远距离质子转移的催化机理的见解，并全面阐明了Nep1的作用方式。
