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.

我的团队正在致力于开发核磁共振辅助晶体学——协同组合 固态核磁共振、X 射线晶体学和计算化学——作为 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 通过定位氢原子，该技术提供了经常缺失的关键化学物质。 连接结构和机制所必需的信息，并为合理的设计提供关键信息。 治疗方法的设计分为三部分：X射线晶体学用于提供粗略的结构。 使用计算化学构建活性位点的化学详细模型的框架， 通过比较可以定量地区分这些模型所探索的各种活性位点化学； 他们预测的 NMR 化学位移与固态 NMR 实验的结果提供了足够的证据。 在活性位点内测量化学位移限制的数量，核磁共振辅助晶体学可以 独特地识别结构。目标系统包括吡哆醛-5’-磷酸 (PLP) 依赖性的。 酶与许多健康状况有关并作为治疗疾病的目标，以及 β-内酰胺酶，介导对β-内酰胺抗生素的抗生素耐药性。 PLP 依赖性酶家族参与氨基酸和其他胺的代谢 这种单一的辅助因子可以参与多种化学转化， 包括外消旋、转氨基、α/β-脱羧、α/β/γ-消除和取代。 了解活性位点如何针对不同的此类反应微调相同的辅因子是主要目标 为了实现这一理解，采用核磁共振辅助晶体学来表征。 在色氨酸合酶中，这些酶促转化具有原子分辨率，这使我们能够进行观察。 反应协调进出多个中间体，这里质子化状态完成了。 例如，化学图解为什么特定抑制剂（如苯并咪唑）无法反应形成 共价键，因为它通过氢键与 βGlu109 和带电的 ε-氨基保持错误的方向 βLys87 组。 第二个目标是扩大在 PLP 依赖性酶促转化表征方面的成功。 从 Toho-1 β-内酰胺酶开始，我们以最初的化学位移为基础。 溶液动力学的分配和表征，以研究用于抑制的化学机制 在此应用中，将在中子界面处开发核磁共振辅助晶体学。 晶体学，迄今为止还无法解析抑制剂存在下的结构，但是 了解化学水平的机制需要我们指定关键的质子化状态 活性位点酸/碱催化残基。