Mutational analysis of TXNRD1 reveals the essential role of Trp in TRP14 reduction and identifies key determinants of enzymatic activity and thermostability.

Cytosolic thioredoxin reductase (TXNRD1) is a key selenoenzyme involved in cellular redox regulation and antioxidant defense. Elucidating the catalytic mechanism of TXNRD1 and its conserved residues or domains is crucial for drug discovery and development. In this study, we investigated the functional roles of several conserved residues in TXNRD1, including Trp, Tyr, and residues in the guiding bar motif, and the catalytic C-terminal domain. Using recombinant TXNRD1 mutants, we found that Trp mutants partially retain TXNRD1 activity in reducing TXN1 or TXNL1 but completely abolish the TRP14 reducing activity. Notably, the function of Trp in TXNRD1 catalysis is independent of the Sec residue and unrelated with the oligomerization status of TXNRD1. AlphaFold3 structural modeling suggested that Trp may form hydrogen bonds with the TXN-like protein, potentially facilitating the approach of the C-terminal tail of TXNRD1 to the buried disulfide bond (Cys-Cys) in TRP14. In contrast, the disulfide bonds of TXN1 and TXNL1 are more accessible, highlighting a unique structural requirement of Trp in TRP14 reduction. Furthermore, we demonstrated that both the presence and its precise positioning of Sec in the C-terminal tail are critical for catalytic activity. Additionally, Asp and Arg were found to play important roles in maintaining TXNRD1 thermostability. Together, our findings underscore the unique role of Trp in modulating TXNRD1's catalytic activity and provide new insights into the regulation of TXNRD1 activity and stability, which deepens our understanding of selenoprotein function and the mechanistic basis of redox regulation.