# Chemically-Rich Structure and Dynamics in the Active Site of Tryptophan Synthase > **NIH NIH R01** · UNIVERSITY OF CALIFORNIA RIVERSIDE · 2020 · $343,556 ## Abstract 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. ## Key facts - **NIH application ID:** 9999607 - **Project number:** 5R01GM097569-09 - **Recipient organization:** UNIVERSITY OF CALIFORNIA RIVERSIDE - **Principal Investigator:** Leonard J Mueller - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $343,556 - **Award type:** 5 - **Project period:** 2011-09-30 → 2022-08-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/9999607 ## Citation > US National Institutes of Health, RePORTER application 9999607, Chemically-Rich Structure and Dynamics in the Active Site of Tryptophan Synthase (5R01GM097569-09). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9999607. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*