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, ?/?-decarboxylation, and ?/?/?- 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 ?-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 ?-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.

Public Health Relevance

Pyridoxal-5?-phosphate (PLP, vitamin-B6)-dependent enzymes catalyze a wide array of transformations required for amino acid metabolism. These ubiquitous enzymes have been implicated in health conditions and as targets for treating diseases such as Tay-Sachs, metachromatic leukodystrophy, and tuberculosis. Understanding the catalytic mechanism of PLP enzymes at the atomic/molecular level will provide the necessary chemical details for developing them as important targets for drug design.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Preusch, Peter
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University of California Riverside
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Wang, Luther; Uribe-Romo, Fernando J; Mueller, Leonard J et al. (2018) Predicting anisotropic thermal displacements for hydrogens from solid-state NMR: a study on hydrogen bonding in polymorphs of palmitic acid. Phys Chem Chem Phys 20:8475-8487
Hilario, Eduardo; Caulkins, Bethany G; Huang, Yu-Ming M et al. (2016) Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water. Biochim Biophys Acta 1864:268-279
Huang, Yu-Ming M; You, Wanli; Caulkins, Bethany G et al. (2016) Protonation states and catalysis: Molecular dynamics studies of intermediates in tryptophan synthase. Protein Sci 25:166-83
Caulkins, Bethany G; Young, Robert P; Kudla, Ryan A et al. (2016) NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity. J Am Chem Soc 138:15214-15226
Chang, Chia-En A; Huang, Yu-Ming M; Mueller, Leonard J et al. (2016) Investigation of Structural Dynamics of Enzymes and Protonation States of Substrates Using Computational Tools. Catalysts 6:
Hartman, Joshua D; Kudla, Ryan A; Day, Graeme M et al. (2016) Benchmark fragment-based (1)H, (13)C, (15)N and (17)O chemical shift predictions in molecular crystals. Phys Chem Chem Phys 18:21686-709
Young, Robert P; Caulkins, Bethany G; Borchardt, Dan et al. (2016) Solution-State (17)O?Quadrupole Central-Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase. Angew Chem Int Ed Engl 55:1350-4
Hartman, Joshua D; Neubauer, Thomas J; Caulkins, Bethany G et al. (2015) Converging nuclear magnetic shielding calculations with respect to basis and system size in protein systems. J Biomol NMR 62:327-40
Caulkins, Bethany G; Yang, Chen; Hilario, Eduardo et al. (2015) Catalytic roles of ?Lys87 in tryptophan synthase: (15)N solid state NMR studies. Biochim Biophys Acta 1854:1194-9
Caulkins, Bethany G; Bastin, Baback; Yang, Chen et al. (2014) Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate. J Am Chem Soc 136:12824-7

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