Chemically-Rich Structure and Dynamics in the Active Site of Tryptophan Synthase

色氨酸合酶活性位点的化学丰富结构和动力学

• 批准号: 9384666
• 负责人: Leonard J Mueller
• 金额: $ 44.76万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9384666
• 关键词: Active Sites; Amination; Amines; Aspartate Transaminase; Catalysis; Charge; Chemicals; Computing Methodologies; Crystallization; Crystallography; Data; Decarboxylation; Diamines; Dimensions; Disease; Drug Design; Drug Targeting; Electrostatics; Environment; Enzymes; Family; Goals; Health; Human; Hybrids; Hydrogen; Indoles; Investigation; Isotope Labeling; Link; Location; Measures; Metachromatic Leukodystrophy; Modeling; Molecular; Mutation; Neutron Diffraction; Nuclear; Organism; Pathway interactions; Play; Positioning Attribute; Process; Protons; Pyridoxal Phosphate; Reaction; Resolution; Roentgen Rays; Role; Sampling; Side; Site; Specific qualifier value; Specificity; Sphingolipids; Structural Models; Structure; Substrate Interaction; Techniques; Test Result; Testing; Therapeutic; Tryptophan Synthase; Tuberculosis; Vitamin B6; Work; X-Ray Crystallography; alpha ketoglutarate; amino acid metabolism; analog; biochemical tools; chemical reaction; cofactor; computational chemistry; deprotonation; design; experimental study; insight; mutant; novel; protonation; racemization; serine containing aminolipid; serine palmitoyltransferase; solid state nuclear magnetic resonance; structural biology; three dimensional structure; transamination

Project Summary This project develops 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 final and often critical missing chemical information necessary to link structure and mechanism, as well as providing crucial information for the rational design of therapeutics. The goal of this work is to understand the molecular basis for reaction specificity in pyridoxal-5'-phosphate (PLP) dependent enzymes, focusing on the PLP-dependent enzyme tryptophan synthase (TS) and related PLP-dependent enzymes serine palmitoyltransferase (SPT) and aspartate aminotransferase (AAT). PLP- dependent enzymes have been implicated in numerous human health conditions and as targets for treating diseases such as Tay-Sachs, metachromatic leukodystrophy, and tuberculosis. 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. For example, the fold type II enzyme TS catalyzes the synthesis of L-Trp from indole and L-Ser, while the fold type I enzyme SPT catalyzes the first step of sphingolipid synthesis in all organisms using the same type of chemical reaction as TS, despite belonging to a different fold type. AAT is also a fold type I enzyme that catalyzes the transformation of L-Asp and α-ketoglutarate to L-Glu; AAT shares many structural similarities with SPT, yet catalyzes a different type of chemical reaction. Understanding how active sites fine-tune the same cofactor for such varied reactions is a primary objective of this proposal. While stereoelectronic contributions play a clear role, the majority of PLP-dependent transformations are initiated by the same α-deprotonation step, so additional reaction specificity must be conferred during subsequent stages. To accomplish this understanding, NMR-assisted crystallography is employed to characterize these enzymatic transformations with atomic resolution. In this approach, X-ray crystallography provides a coarse framework upon which chemically-rich models of the active site can be developed using computational chemistry, and these models can be distinguished by comparison of their first- principles predicted NMR chemical shifts with the results of SSNMR experiments. Conceptually, each technique is a piece of a larger puzzle that when solved provides an unprecedented view of enzyme catalysis.

项目摘要 该项目开发NMR辅助结晶学-固态的协同组合 核磁共振、X射线晶体学和计算化学--作为原子-- 酶活性位点的分辨探针，能够确定所有原子的位置，包括 氢。通过定位氢原子，这项技术提供了最终的，往往是关键的失踪化学品， 联系结构和机制所需的信息，以及为合理的 治疗设计。 这项工作的目标是了解吡哆醛-5 '-磷酸反应特异性的分子基础 (PLP)依赖酶，重点是PLP依赖酶色氨酸合成酶（TS）和相关 PLP依赖性酶丝氨酸棕榈酰转移酶（SPT）和天冬氨酸转氨酶（AAT）。PLP- 依赖性酶与许多人类健康状况有关， 疾病如泰-萨二氏病、异染性脑白质营养不良和结核病。依赖PLP的家庭 酶参与氨基酸和其它含胺生物分子的代谢。这间单人 辅因子可参与多种化学转化，包括外消旋化，转氨作用， α/β-脱羧和α/β/γ-消除和取代。例如，折叠II型酶TS 催化从吲哚和L-Ser合成L-Trp，而折叠I型酶SPT催化第一个 在所有生物体中使用与TS相同类型的化学反应进行鞘脂合成的步骤，尽管 属于不同的褶皱类型。AAT也是催化L-Asp转化的折叠I型酶 和α-酮戊二酸转化为L-Glu; AAT与SPT具有许多结构相似性，但催化不同类型的 化学反应 了解活性位点如何微调相同的辅因子，以适应不同的反应是一个主要目标 这一提议。虽然立体电子贡献发挥了明显的作用，但大多数PLP依赖性 转化是由相同的α-去质子化步骤引发的，因此必须考虑额外的反应特异性。 在后续阶段授予。为了实现这一理解，NMR辅助结晶学是 采用原子分辨率来表征这些酶促转化。在这种方法中，X射线 晶体学提供了一个粗略的框架，在其上可以 使用计算化学开发，这些模型可以通过比较它们的第一个- 用SSNMR实验结果预测了NMR化学位移。从概念上讲，每个 技术是一个更大的难题的一部分，当解决了酶催化提供了一个前所未有的看法。