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.

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Sugar alcohols (polyols) are abundant carbohydrates in lichen-forming algae and transported to other lichen symbionts, fungi, and bacteria. Particularly, ribitol is an abundant polyol in the lichen Cetraria sp. Polyols have important physiological roles in lichen symbiosis, but polyol utilization in lichen-associated bacteria has been largely unreported. Herein, we purified and characterized a novel ribitol dehydrogenase (RDH) from a Cetraria sp.-associated bacterium Sphingomonas sp. PAMC 26621 grown on a minimal medium containing D-ribitol (the RDH hereafter referred to as SpRDH). SpRDH is present as a trimer in its native form, and the molecular weight of SpRDH was estimated to be 39 kDa by SDS-PAGE and 117 kDa by gel filtration chromatography. SpRDH converted D-ribitol to D-ribulose using NAD + as a cofactor. As far as we know, SpRDH is the first RDH belonging to the medium-chain dehydrogenase/reductase family. Multiple sequence alignments indicated that the catalytic amino acid residues of SpRDH consist of Cys37, His65, Glu66, and Glu157, whereas those of short-chain RDHs consist of Ser, Tyr, and Lys. Furthermore, unlike other short-chain RDHs, SpRDH did not require divalent metal ions for its catalytic activity. Despite SpRDH originating from a psychrophilic Arctic bacterium, Sphingomonas sp., it had maximum activity at 60˚C and exhibited high thermal stability within the 4-50˚C range. Further studies on the structure/function relationship and catalytic mechanism of SpRDH will expand our understanding of its role in lichen symbiosis.