# Structural and proton dynamics of pyridoxal-5'-phosphate dependent enzymes (resubmission)

> **NIH NIH R01** · UNIVERSITY OF GEORGIA · 2021 · $622,775

## Abstract

Enzymes containing pyridoxal-5'-phosphate (PLP) are involved in a broad range of reactions of amino
acids and amines, including transamination, racemization, decarboxylation, β- and γ-elimination, β- and γ-
substitution, and, as recently discovered, even oxidation and oxygenation. A number of important current or
prospective drug targets are PLP-dependent enzymes, including γ-aminobutyrate aminotransferase, DOPA
decarboxylase, alanine racemase, ornithine decarboxylase, and serine hydroxymethyltransferase. However,
many of the current drugs that target PLP-dependent enzymes suffer from side effects due to lack of specificity
for their targets. Thus, it is important to understand the reactions of these enzymes with molecular and atomic
levels of detail to help in the design of new more potent and more selective drugs. Using X-ray crystallography,
a great deal has been learned about the role of both enzymes and cofactor in catalysis. Despite this, there are still
critical gaps in our understanding of PLP-dependent enzymes that limit drug design. Crystal structures alone are
missing two essential pieces of information. First, they lack important information regarding reaction dynamics.
Protein motion in ligand binding and catalysis is known to play a central role in enzymes, but how this occurs is
essentially unknown. In addition, hydrogen atoms that play critical roles in PLP catalysis are not directly
observed by X-ray crystallography. This leaves a significant gap in our understanding of general acid-base
catalysis in enzymes in general and particularly in PLP-dependent enzymes, where active site protonation states
appear to play critical roles in control of reaction specificity. A recent neutron diffraction structure of aspartate
aminotransferase found a proton in an unpredicted position in the active site, forming a low barrier hydrogen
bond between the substrate carboxylate and the aldimine nitrogen. This void in our understanding of protonation
and ionization states impedes rational design of therapeutic agents that, for example, are tailored for specific
electrostatic environments. The goal of the proposed project is to provide a very detailed understanding of PLP
enzyme mechanisms by coordinately defining their structures and dynamics from the global to the atomic level.
To accomplish this, we will employ a synergistic combination of biophysical techniques that are sensitive to
different size- and time-scales. These will include joint X-ray/neutron crystallography, solid-state NMR
crystallography, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM)
calculations, inelastic neutron scattering, steady-state and rapid kinetics techniques of PLP dependent enzymes.
The results of this collaborative venture will provide, for the very first time, a global picture of catalysis by a
large and centrally important class of enzymes at true atomic-resolution for stable intermediates as well as the
dynamic connections between them. Th...

## Key facts

- **NIH application ID:** 10264149
- **Project number:** 5R01GM137008-02
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** Andrii Y Kovalevskyi
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $622,775
- **Award type:** 5
- **Project period:** 2020-09-15 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10264149

## Citation

> US National Institutes of Health, RePORTER application 10264149, Structural and proton dynamics of pyridoxal-5'-phosphate dependent enzymes (resubmission) (5R01GM137008-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10264149. Licensed CC0.

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