Department of Physical Chemistry, Faculty of Science, Palacky University, 17. Listopadu 12, 771 46 Olomouc, Czech Republic.
J Phys Chem B. 2010 Jul 8;114(26):8701-12. doi: 10.1021/jp9109699.
The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, the detailed reaction mechanism as well as the protonation state of the glmS reactive form still remains elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the Thermoanaerobacter tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40(-) seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase in K(m) with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. On the basis of the identified dominant protonation state of the reaction precursor, we propose a hypothesis of the self-cleavage mechanism in which A-1(2'-OH) is activated as a nucleophile by the G1(pro-R(p)) nonbridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group.
glmS 催化型核糖开关是编码葡萄糖胺-6-磷酸（GlcN6P）合酶（glmS）的 mRNA5'-非翻译区的一部分，在许多革兰氏阳性菌中都存在。该 RNA 能与辅助因子 GlcN6P 结合，并诱导 RNA 的位点特异性自我切割。然而，详细的反应机制以及 glmS 反应形式的质子化状态仍然难以捉摸。为了探测关键活性位点残基的主要质子化状态，我们进行了涉及三种关键活性位点部分的各种质子化状态的明确溶剂分子动力学模拟，这些部分在现有晶体结构中观察到：（i）G40（根据 Thermoanaerobacter tengcongensis 编号），（ii）GlcN6P 的氨基/铵基，和（iii）GlcN6P 的磷酸部分。我们发现，去质子化的 G40(-)似乎与观察到的 glmS 活性位点结构不兼容。我们的数据表明，G40 的典型形式通过稳定切割位点 A-1(2'-OH)亲核试剂的直线攻击构象，而不是更直接的化学作用，发挥结构作用。此外，我们观察到 GlcN6P 磷酸部分质子化会导致辅助因子结合减弱，这解释了实验观察到的 K(m)随 pH 值降低而增加的现象。最后，我们讨论了辅助因子结合及其与 G65 和 G1 嘌呤的相互作用在 A-1(2'-OH)直线攻击构象的结构稳定中的可能作用。基于确定的反应前体的主要质子化状态，我们提出了一种自我切割机制的假设，其中 A-1(2'-OH)被裂解磷酸盐的 G1(pro-R(p))非桥氧原子激活为亲核试剂，而 GlcN6P 的铵基则作为质子化 G1(O5')离去基团的广义酸。
