Robust structural, kinetic and biophysical characterization of wild-type human ACOD1, selected mutants and their interaction with citraconate
TL;DR
This work presents a thorough structural investigation that yielded the first ligand-bound structure of this protein family, which required the generation of artifact-free apo crystals, and developed a novel, low-consumption, robust kinetic assay.
Abstract
Aconitate decarboxylase 1, an enzyme member of the MmgE-PrpD family of proteins, has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases. Its product, itaconate, is a multifunctional metabolite shown to drive several disease states. Though extensively studied in cellulo and in vivo, this protein is biochemically and mechanistically under characterized and although a family of inhibitors has been described, no ligand-bound structures have yet been determined. In this work we present a thorough structural investigation that yielded the first ligand-bound structure of this protein family, which required the generation of artifact-free apo crystals. We also developed a novel, low-consumption, robust kinetic assay and investigated active site and allosteric mutants to further elucidate structural and dynamic activity relationships of this protein.