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

项目摘要 该项目开发了核磁共振辅助结晶学-固态的协同组合 核磁共振、X射线结晶学和计算化学--作为一种原子-- 酶活性部位的分辨探针，能够确定所有原子的位置，包括 氢气。通过定位氢原子，这项技术提供了最终也往往是关键的丢失的化学物质 链接结构和机制所需的信息，以及为Rational 治疗学的设计。 本工作的目的是了解吡哆醛-5‘-磷酸反应专一性的分子基础。 (PLP)依赖酶，重点是PLP依赖酶色氨酸合成酶(TS)和相关 依赖PLP的丝氨酸棕榈酰转移酶(SPT)和天冬氨酸氨基转移酶(AAT)。PLP- 依赖酶与许多人类健康状况有关，也是治疗的靶点。 疾病，如泰-萨克斯病、异色性脑白质营养不良症和肺结核。PLP依赖型家族 酶参与氨基酸和其他含胺生物分子的新陈代谢。这首单曲 辅因子可以参与一系列不同的化学转化，包括外消旋，转氨基， α/β-脱羧基，α/β/γ-消除和取代。例如，折叠II型酶TS 催化由吲哚和L-丝氨酸合成L-色氨酸，而折叠I型酶催化第一个 在所有生物体中使用与TS相同类型的化学反应合成鞘磷脂的步骤，尽管 属于不同的褶皱类型。AAT也是一种I型折叠酶，催化L-天冬氨酸的转化 和α-酮戊二酸到L-Glu；AAT在结构上与SPT有许多相似之处，但催化了一种不同类型的 化学反应。 了解活性中心如何针对不同的反应微调相同的辅因子是一个主要目标 这项提议。虽然立体电子贡献起着明显的作用，但大多数依赖于PLP的 转化是由相同的α去质子化步骤启动的，因此额外的反应特异性必须是 在随后的阶段授予的。为了实现这一理解，核磁共振辅助结晶学是 用来用原子分辨率来表征这些酶转化。在这种方法中，X射线 结晶学提供了一个粗略的框架，在该框架上可以建立富含化学成分的活性中心模型 使用计算化学开发的，这些模型可以通过比较它们的第一个- 原理用SS核磁共振实验结果预测了核磁共振化学位移。从概念上讲，每个 技术是一个更大的谜题中的一块，当它被解决时，它提供了关于酶催化的前所未有的观点。

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.