Defining Factors that Influence the Stability of Tetrameric EgtB from <i>Chloracidobacterium thermophilum</i>: From pH Modulation to Affinity Tag Retention

Ergothioneine (EGT) is a vital antioxidant synthesized exclusively by microorganisms and is associated with various oxidative stress-related diseases in humans. EGT biosynthesis is catalyzed by a gene cluster containing the enzyme EgtB, a nonheme iron-dependent sulfoxide synthase. While multiple EgtB enzymes have been characterized, EgtB from <i>Chloracidobacterium thermophilum</i> (<i>Cth</i>) uniquely forms a homotetrameric structure, in contrast to the monomeric forms of its homologues. In this study, we applied biophysical and biochemical techniques to examine the secondary and quaternary structures of metal-free (apo) <i>Cth</i>EgtB, focusing on how pH modulation and thermodynamic factors influence its stability and oligomerization. Herein, we show that key factors such as pH, temperature, and the presence of a 6xHistidine tag impact the enzyme's structural integrity and thermal stability. This work underscores the importance of considering structural and environmental factors of sulfoxide synthases to alter or improve their catalytic efficiency.