Flexibility in acidophilic thioredoxins: Insights from Asp43 substitutions in E. coli thioredoxin.

Thioredoxins (Trxs) are small, highly conserved oxidoreductases characterized by a redox-active CXXC motif. While the structural adaptation of modern Trxs such as Escherichia coli Trx (EcTrx) has been described, the mechanisms underlying Trx adaptation to acidic environments remain unclear. In EcTrx, Asp43 stabilizes the active-site region through a water-mediated hydrogen bond with Lys57, which modulates the protonation state of Cys32 in cooperation with Asp26. Comparative sequence analysis shows that small, nonpolar amino acids-such as Gly and Ala-are frequently found at position 43 in acidophilic Trxs, indicating that structural flexibility at this position may contribute to acidic adaptation. To test how substitutions at position 43 affect Trx stability and function, we generated EcTrx mutants (D43G, D43A, D43S, D43N, D43L, and D43E). Thermal shift and guanidinium chloride-induced unfolding assays revealed that D43A and D43S markedly reduced stability, while D43G retained moderate stability, likely due to tighter N-terminal packing as observed in the acidophilic Acetobacter aceti Trx. These three mutants also displayed increased conformational flexibility, whereas D43N and D43L conferred partial stabilization through polar or hydrophobic side chains. In contrast, D43E-mimicking ancestral Glu-restored hydrogen bonding and enhanced thermal stability, resulting in the most rigid structure. Most mutants retained catalytic activity in DTNB assays, except D43S, which showed 50% of wild-type activity. Overall, our results demonstrate that Asp43 is critical for maintaining EcTrx structural stability and suggest that enhanced, but not excessive, flexibility at this position facilitates Trx adaptation to acidic environments.