NMR crystallography: Imaging active site chemistry and protonation states

NMR 晶体学：对活性位点化学和质子化状态进行成像

• 批准号: 10406831
• 负责人: Leonard J Mueller
• 金额: $ 37.82万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10406831
• 关键词: Acids; Active Sites; Amines; Antibiotic Resistance; Antibiotics; Antidiabetic Drugs; Antimalarials; Cephalosporins; Charge; Chemicals; Chemistry; Crystallography; Decarboxylation; Disease; Drug Design; Enzymes; Family; Goals; Health; Hydrogen; Hydrogen Bonding; Image; Investigation; Link; Measures; Mediating; Metachromatic Leukodystrophy; Modeling; Molecular; Monobactams; Neutrons; Parkinson Disease; Penicillins; Pharmaceutical Preparations; Positioning Attribute; Pyridoxal Phosphate; Reaction; Resolution; Structure; System; Techniques; Therapeutic; Tryptophan Synthase; Tuberculosis; Vitamin B6; Work; X-Ray Crystallography; amino acid metabolism; amino group; base; benzimidazole; beta-Lactam Resistance; beta-Lactamase; cofactor; computational chemistry; covalent bond; enzyme mechanism; experimental study; improved; inhibitor; novel; peer; protonation; racemization; rational design; restraint; solid state nuclear magnetic resonance; success; transamination

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.

我的小组正在努力发展NMR辅助晶体学 - 固态核磁共振，X射线晶体学和计算化学 - 作为一种 酶活性位点的原子分辨率探针，能够定义所有原子的位置，包括 氢。通过定位氢原子，该技术提供了通常关键的缺失化学物质 链接结构和机制所需的信息，并为理性提供关键信息 该方法是三倍：X射线晶体学用于提供粗糙的结构 使用计算化学的化学位点的化学详细模型的框架， 并探索了各种活跃的现场化学；这些模型可以通过比较来定量区分 他们预测的NMR化学移位是固态NMR实验的结果。提供了足够的 化学位移约束数量是在活性部位中测量的，NMR辅助晶体学可以 独特地识别结构。靶向系统包括吡啶还原-5'-磷酸（PLP）依赖性 酶，这些酶在许多健康状况中被暗示，作为治疗疾病的靶标，并且 β-内酰胺酶，中位对β-内酰胺抗生素的抗生素抗性。 PLP依赖性酶的家族参与氨基酸和其他胺的代谢 含有生物分子。这个单个辅因子可以参与一系列的化学转化阵列， 包括种族化，跨跨，α/β-二羧化以及α/β/γ-脱位和取代。 了解这种不同反应的主动位点如何微调相同的辅助因子是 这个建议。为了实现这一理解，进行NMR辅助晶体学以表征 这些具有原子分辨率的酶转化。在色氨酸合酶中，这使我们可以前 反应坐在多个中间体中坐标。质子化状态在这里完成 化学图片，例如为什么特定的抑制剂（例如苯依咪唑）无法做出反应以形成 通过氢键与βGlu109和带电的ε-氨基键在错误的方向上保持共价键 一组βLys87。 第二个目标是扩展表征PLP依赖性酶促转化的成功 从TOHO-1β-内酰胺酶开始的酶至β-内酰胺酶。在这里，我们以最初的化学转移为基础 解决方案中动力学的分配和表征，以研究用于抑制的化学机制 抗生素。在此应用中，将在与中子的接口处开发NMR辅助晶体学 晶体学，迄今为止无法在存在抑制剂的情况下解决结构，但是 了解化学水平的机制要求我们分配密钥的质子化状态 活性位点酸/碱催化残留物。