Abstract
Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central β-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the β2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures.
Highlights
Thioredoxin (Trx) is a class of small redox proteins (~12 kDa) that protect cells against oxidative stress [1, 2]
Since F26 was highly conserved in Trxs from diverse working temperatures and positioned in a hydrophobic environment, this study examined the effects of F26 mutations, at the aromatic residues and the smallest hydrophobic residue, on the cold adaptation of SpTrx, its interaction with SpTR, and oxidoreductase activity
The structural model of SpTrx based on the crystal structure of mesophilic Acetobacter aceti Trx (54.2% amino acid sequence identity) showed a typical Trx fold with five β-strands sandwiched on both sides by a pair of α-helices (Fig 1A)
Summary
Thioredoxin (Trx) is a class of small redox proteins (~12 kDa) that protect cells against oxidative stress [1, 2]. The reduced Trx passes electrons to many cytoplasmic proteins, including ribonucleotide reductase [5] and methionine sulfoxide reductase [6]. Buried F26 is important for the structural stability of psychrophilic SpTrx fold—four β-strands surrounded by three α-helices [2]. The sequence reconstruction of ancestral thioredoxins (Trxs), which goes back approximately four billion years, shows a robust structure and high thermal stability [9, 10]
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