# Expected and Unexpected “Guests” at the Active Site of Human Orotidine 5′-Monophosphate Decarboxylase

## TL;DR

This study explores the active site of the highly efficient enzyme OMPDC using X-ray crystallography and binding experiments to understand how different molecules interact with it.

## Contribution

The study identifies unexpected ligands at the active site of OMPDC and proposes new strategies for designing more effective inhibitors.

## Key findings

- XMP and dTMP were found bound to OMPDC and its Thr321Asn variant with high binding affinities.
- A new transition-state analogue, YMP, formed a favorable hydrogen bond but faced an entropic penalty.
- 5-methyl OMP improved ligand-enzyme interactions through hydrophobic stabilization.

## Abstract

With an extraordinary
rate enhancement of 1017 compared
to the uncatalyzed reaction and no need for a cofactor, orotidine
5′-monophosphate decarboxylase (OMPDC) is considered one of
the most efficient enzymes. Its mechanism has fascinated researchers
for over 50 years. In this study, we used high-resolution X-ray crystallography
to examine the molecular interactions between the active site of human
OMPDC and various natural and synthetic ligands, including transition-state
and product analogues, at the atomic level. Additionally, we evaluated
their binding affinities with isothermal titration calorimetry (ITC).
During protein expression and subsequent structure analysis, we identified
nucleotides xanthosine-5′-monophosphate (XMP) and thymidine-5′-monophosphate
(dTMP) bound to the active sites of OMPDC and its Thr321Asn variant,
respectively, and confirmed their high binding affinities through
ITC. Chemically, we investigated the role of the ribose 2′–OH
group using 2′-deoxy OMP and 2′-SH UMP, focusing on
validating key binding interactions within the nucleoside moiety.
To further explore these interactions, we modified the heterocycles
(e.g., GMP and CMP) and synthesized a new transition-state analogue,
cyanuryl-5′-monophosphate (YMP). YMP exhibited strong affinity
for OMPDC and formed an additional hydrogen bond with a nearby water
molecule. However, this enthalpically favorable interaction resulted
in an entropic penalty compared to the best-known OMPDC inhibitor,
BMP, leading to similar affinities. To address this, we synthesized
5-methyl OMP to further improve ligand-enzyme interactions. This modification
enhanced stabilization within the hydrophobic pocket through van der
Waals forces, paving the way for designing more effective OMPDC inhibitors
with specific substitutions aimed at optimizing binding affinity and
enzyme inhibition.