# Structural and Biophysical Analyses of Human Prostamide/Prostaglandin F Synthase with Two Active Form-Mimicking Mutations

## TL;DR

This paper reveals the structure and function of human PGFS, a redox-regulated enzyme involved in regulating intraocular pressure and labor.

## Contribution

The study provides the first crystal structure of human PGFS with active site mutations and reveals its unique structural and biophysical features.

## Key findings

- The PGFS double mutant adopts a thioredoxin-like fold with a flexible Tyr108–Asp124 region.
- Pro167 in PGFS adopts a trans-conformation, influencing the localization of Arg40 and creating a positive charge near the CXXC motif.
- Reduction of the CXXC disulfide bond increases PGFS thermal stability but also induces structural disorder upon ligand binding.

## Abstract

Human prostamide/prostaglandin F synthase (PGFS) catalyzes the NADPH-dependent conversion of prostaglandin H2 (PGH2) to prostaglandin F2α that plays a key role in regulating intraocular pressure and labor. Despite its physiological importance, structural and biochemical information of the human PGFS has been limited because of difficulties in obtaining sufficient quality of PGFS wild-type crystal and short half-life of PGH2. Here, we report the crystal structure of human PGFS with two active site mutations, C44S/C47S double mutant (DM), which mimics the reduced active form of the CXXC motif of human PGFS. Structural analysis revealed that PGFS DM adopts a typical thioredoxin (Trx)-like fold. Analysis of B-factors and MD simulations reveals that Tyr108–Asp124 is an intrinsically flexible region, devoid of any stabilizing crystal contacts. Unlike canonical Trx-like proteins, Pro167 in PGFS adopts a trans-conformation, inducing a specific Arg40 side chain localization that creates a positive charge near the CXXC motif. Activation of PGFS by reduction of disulfide bond in the CXXC motif enhanced the thermal stability via core stabilization, yet an unexpected increase in the structural disorder was detected with CD spectroscopy, especially upon ligand binding. These findings collectively establish PGFS as a structurally distinct and redox-regulated enzyme. Our results provide novel molecular insights into PGFS as an underexplored but promising therapeutic target.