Trp101-Mediated Cold Adaptation in <i>Sphingomonas</i> sp. Thioredoxin: Increased α4-Helix Rigidity with Preserved Overall Flexibility

Thioredoxin (Trx) is a small, conserved redox protein composed of five β-strands and four α-helices, and it reduces disulfide bonds and helps maintain cellular redox homeostasis. During evolution from the Trx of the last bacterial common ancestor (LBCA Trx) to <i>Escherichia coli</i> Trx (EcTrx), the α3 helix became more flexible, while the α4 helix gained rigidity. To investigate the role of α4-helix rigidity in cold adaptation, we analyzed <i>Sphingomonas</i> sp. Trx (SpTrx), a cold-adapted ortholog, focusing on α2-α4 salt bridges and α4-β5 hydrophobic interactions. We constructed single and double mutants, targeting Glu43, Glu47, and Trp101, and assessed structural stability using thermal shift assays, chemical denaturation, fluorescence spectroscopy, and circular dichroism. Catalytic activity was evaluated using insulin reduction and DTNB-based kinetic assays. Salt bridge mutations (E43A, E47A, and E43A/E47A) modestly decreased both stability and enzyme activity. In contrast, hydrophobic interface mutations (W101A and W101F) caused more substantial destabilization, with W101A inducing the most pronounced structural disruption. The E47A/W101F double mutant exhibited poor expression and the lowest stability and activity. In a comparative study with EcTrx, salt bridge mutations had a greater impact on thermal stability than in SpTrx. While F102A significantly reduced stability and increased flexibility, F102W (the SpTrx-equivalent substitution) increased both stability and rigidity. These findings demonstrate that in SpTrx, Trp101 enhances α4-helix rigidity through hydrophobic packing while preserving overall flexibility. This study highlights the evolutionary divergence of stabilization mechanisms in Trx orthologs and provides insight into how cold-adapted enzymes maintain activity at low temperatures.