NMR crystallography: Imaging active site chemistry and protonation states

我的团队正在开发核磁共振辅助结晶学-协同组合 固态核磁共振，X射线晶体学和计算化学-作为一个 酶活性位点的原子分辨率探针，能够确定所有原子的位置，包括 氢。通过定位氢原子，这项技术提供了通常关键的化学物质， 联系结构和机制所需的信息，以及为合理的 治疗设计。该方法是三重的：X射线晶体学用于提供粗略的结构 使用计算化学建立活性位点的化学详细模型的框架， 和各种活性中心化学探索;这些模型可以定量区分，通过比较 他们预测的NMR化学位移与固态NMR实验的结果。提供了充分 在活性位点内测量许多化学位移限制，NMR辅助结晶学可以 唯一标识结构。靶向系统包括吡哆醛-5 '-磷酸（PLP）依赖性 酶，其与许多健康状况有关，并作为治疗疾病的靶点， β-内酰胺酶，其介导对β-内酰胺抗生素的抗生素抗性。 PLP依赖性酶家族参与氨基酸和其他胺的代谢， 含有生物分子。这种单一的辅因子可以参与多种多样的化学转化， 包括外消旋化、转氨作用、α/β-脱羧作用和α/β/γ-消除和取代。 了解活性位点如何微调这种不同反应的相同辅因子是一个主要目标， 这个提议。为了实现这一理解，NMR辅助结晶学被用来表征 这些酶促转化与原子分辨率。在色氨酸合成酶中，这使我们能够沿着 反应协调进入和离开多个中间体。在这里质子化状态完成 化学图片，例如，为什么特定的抑制剂，如苯并咪唑不能反应，形成一个 共价键，因为它通过氢键与β Glu 109和带电荷的ε-氨基保持错误的方向 β Lys 87组。 第二个目标是扩展在表征PLP依赖性细胞中的酶促转化方面的成功。 从Toho-1 β-内酰胺酶开始。在这里，我们建立在我们最初的化学位移 分配和表征溶液中的动力学，以研究用于抑制 抗生素在这个应用中，NMR辅助结晶学将在与中子的界面处发展。 晶体学，迄今为止还无法解决抑制剂存在下的结构，但其中 要在化学水平上理解这一机制，我们需要确定键的质子化状态， 活性位点酸/碱催化残基。

My group is working to develop NMR-assisted crystallography – the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry – as an atomic-resolution probe of enzyme active sites, capable of defining the position of all atoms, including hydrogens. By locating hydrogen atoms, this technique provides the often critical missing chemical information necessary to link structure and mechanism, as well as providing crucial information for the rational design of therapeutics. The approach is three-fold: X-ray crystallography is used to provide a coarse structural framework upon which chemically-detailed models of the active site are built using computational chemistry, and various active site chemistries explored; these models can be quantitatively distinguished by comparing their predicted NMR chemical shifts with the results from solid-state NMR experiments. Provided a sufficient number of chemical shift restraints are measured within the active site, NMR-assisted crystallography can uniquely identify the structure. The targeted systems include pyridoxal-5’-phosphate (PLP)-dependent enzymes, which have been implicated in numerous health conditions and as targets for treating diseases, and the β-Lactamases, which mediate antibiotic resistance to β-lactam antibiotics. The family of PLP-dependent enzymes are involved in the metabolism of amino acids and other amine- containing biomolecules. This single cofactor can participate in a diverse array of chemical transformations, including racemization, transamination, α/β-decarboxylation, and α/β/γ- elimination and substitution. Understanding how active sites fine-tune the same cofactor for such varied reactions is a primary objective of this proposal. To accomplish this understanding, NMR-assisted crystallography is employed to characterize these enzymatic transformations with atomic resolution. In tryptophan synthase, this allows us to peer along the reaction coordinates into and out of multiple intermediates. Here the protonation states complete the chemical picture for why, for example, specific inhibitors such as benzimidazole are unable to react to form a covalent bond as it is held in the wrong orientation by hydrogen bonds to βGlu109 and the charged ε-amino group of βLys87. A second goal is to extend the successes in characterizing enzymatic transformations in PLP-dependent enzymes to the β-lactamases, starting with the Toho-1 β-lactamase. Here we build on our initial chemical shift assignments and characterization of dynamics in solution to study the chemical mechanism used to inhibit antibiotics. In this application, NMR-assisted crystallography will be developed at the interface with neutron crystallography, which to date has been unable to solve the structure in the presence of an inhibitor, but where understanding the mechanism at the chemical level requires that we assign the protonation states of the key active site acid/base catalytic residues.