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Trp101-Mediated Cold Adaptation in Sphingomonas sp. Thioredoxin: Increased α4-Helix Rigidity with Preserved Overall
Mohammed Shazaly A Elhassan1, Hoa Nguyen1, ChangWoo Lee1
1Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University, Gyeongsan 38453, South Korea.
ACS Omega
|November 24, 2025
Summary
Investigating thioredoxin (Trx) cold adaptation, researchers found that specific hydrophobic interactions, particularly involving Trp101 in Sphingomonas sp. Trx, enhance α4-helix rigidity and enzyme stability at low temperatures.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Thioredoxin (Trx) is a crucial redox protein maintaining cellular homeostasis.
- Evolutionary changes in Trx structure, specifically α-helix flexibility and rigidity, are linked to adaptation.
- Understanding these changes is key to comprehending enzyme function across different temperatures.
Purpose of the Study:
- To investigate the role of α4-helix rigidity in the cold adaptation of *Sphingomonas* sp. Trx (SpTrx).
- To analyze the impact of specific salt bridges and hydrophobic interactions on SpTrx stability and activity.
- To compare stabilization mechanisms between cold-adapted SpTrx and *Escherichia coli* Trx (EcTrx).
Main Methods:
- Construction and analysis of single and double mutants targeting key residues (Glu43, Glu47, Trp101) in SpTrx.
- Assessment of protein structural stability using thermal shift assays, chemical denaturation, fluorescence spectroscopy, and circular dichroism.
- Evaluation of catalytic activity via insulin reduction and DTNB-based kinetic assays.
Main Results:
- Salt bridge mutations (E43A, E47A) moderately reduced SpTrx stability and activity.
- Hydrophobic interface mutations (W101A, W101F) caused significant destabilization, with W101A having the most pronounced effect.
- Mutations mimicking *Escherichia coli* Trx substitutions revealed differences in thermal stability and rigidity, highlighting distinct evolutionary strategies.
Conclusions:
- Trp101 in SpTrx enhances α4-helix rigidity via hydrophobic packing, crucial for maintaining flexibility and activity in cold conditions.
- Evolutionary divergence in stabilization mechanisms contributes to the functional adaptation of Trx orthologs.
- This research provides insights into how cold-adapted enzymes function at low temperatures.
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