# Structural and proton dynamics of pyridoxal-5’-phosphate dependent enzymes Resubmission (Diversity Supplement)

> **NIH NIH R01** · UNIVERSITY OF GEORGIA · 2021 · $11,725

## Abstract

Project Summary
(NO CHANGE IN THE SCOPE OF THE PARENT PROJECT)
PLP-dependent enzymes represent about 2% of the proteome, and a number of them are current or potential
drug targets. There are four major families of pyridoxal-5’-phosphate (PLP)-dependent enzymes, distinguished
by different three-dimensional folds: the aspartate aminotransferase or α-family (Fold I), the tryptophan
synthase or β-family (Fold II), the alanine racemase family (Fold III), and the D-amino acid aminotransferase
family (Fold IV). Using X-ray crystallography, a great deal has been learned about the role of both these
enzymes and cofactor in catalysis. Despite this, there are still critical gaps in our understanding that limit drug
design. The goal of the proposed project is to provide a very detailed understanding of PLP-dependent
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
spectroscopy, molecular dynamics and QM/MM calculations, inelastic neutron scattering, rapid kinetics
techniques, and heavy enzyme kinetic isotope effects. We will focus on four structurally well-characterized
PLP-dependent enzymes, aspartate aminotransferase, serine hydroxymethyltransferase, tyrosine phenol-lyase
and tryptophan synthase, but for which information on protonation and dynamics is lacking. The enzymes are
drug targets (aspartate aminotransferase, serine hydroxymethyltransferase) or serve as models for drug
targets (tryptophan synthase, tyrosine phenol-lyase). These enzymes catalyze diverse reactions, but use the
same cofactor in similar active sites. Thus, we postulate that the reaction specificity must be controlled by a
combination of protein dynamics and selective protonation of reaction intermediates. Joint X-ray/neutron
crystallography will be the foundation of our research, providing an atomic-level structural basis for protein
dynamics and accurate visualization of hydrogen atoms in protein structures at moderate resolutions. The
results of X-ray/neutron crystallography will be combined with novel solid-state NMR crystallography and with
inelastic neutron scattering to characterize the global and local motions of these enzymes at picosecond-to-
microsecond time scales. Pressure-jump relaxation kinetics, and heavy enzyme kinetic isotope effects will
complement and provide dynamic information on domain motion in the picosecond to minute time-scales. It
should be noted that the inelastic neutron scattering, pressure-jump and heavy enzyme kinetics are
complementary techniques in that they all are sensitive to changes in the vibrational motions of the enzyme,
but interrogate at different time scales. All these results will be integrated with molecular computations to
provide an unprecedented complete picture of t...

## Key facts

- **NIH application ID:** 10359304
- **Project number:** 3R01GM137008-01A1S1
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** Leonard J Mueller
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $11,725
- **Award type:** 3
- **Project period:** 2020-09-15 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10359304

## Citation

> US National Institutes of Health, RePORTER application 10359304, Structural and proton dynamics of pyridoxal-5’-phosphate dependent enzymes Resubmission (Diversity Supplement) (3R01GM137008-01A1S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10359304. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*