Abstract
Thioredoxin (Trx) is a conserved, cytosolic reductase in all known organisms. The enzyme receives two electrons from NADPH via thioredoxin reductase (TrxR) and passes them on to multiple cellular reductases via disulfide exchange. Despite the ubiquity of thioredoxins in all taxa, little is known about the functions of resurrected ancestral thioredoxins in the context of a modern mesophilic organism. Here, we report on functional in vitro and in vivo analyses of seven resurrected Precambrian thioredoxins, dating back 1–4 billion years, in the Escherichia coli cytoplasm. Using synthetic gene constructs for recombinant expression of the ancestral enzymes, along with thermodynamic and kinetic assays, we show that all ancestral thioredoxins, as today's thioredoxins, exhibit strongly reducing redox potentials, suggesting that thioredoxins served as catalysts of cellular reduction reactions from the beginning of evolution, even before the oxygen catastrophe. A detailed, quantitative characterization of their interactions with the electron donor TrxR from Escherichia coli and the electron acceptor methionine sulfoxide reductase, also from E. coli, strongly hinted that thioredoxins and thioredoxin reductases co-evolved and that the promiscuity of thioredoxins toward downstream electron acceptors was maintained during evolution. In summary, our findings suggest that thioredoxins evolved high specificity for their sole electron donor TrxR while maintaining promiscuity to their multiple electron acceptors.
Highlights
Thioredoxin (Trx) is a conserved, cytosolic reductase in all known organisms
Previous studies on Precambrian thioredoxins focused on seven resurrected ancestral members dating between 1.4 and 4 billion years, namely the variants Last Bacterial Common Ancestor (LBCA), Last Archaeal Common Ancestor (LACA), Archaeal–Eukaryotic Common Ancestor (AECA), Last Eukaryotic Common Ancestor (LECA), Last Animalia and Fungi Common Ancestor (LAFCA), Last Common Ancestor of the Cyanobacterial, Deinococcus, and Thermus group (LPBCA), and Last ␥-Proteobacteria Common Ancestor (LGPCA) [15]
The results revealed a clear correlation between the activities of the ancestral thioredoxins in catalyzing the reduction of S-methionine sulfoxide (MetO) by NADPH–reduced tetra(cyclohexylammonium) salt (NADPH) (Fig. 5) and their ability to interact with E. coli thioredoxin reductase (ecTrxR) (Fig. 6a and Table 3)
Summary
Thioredoxin; TNB, thionitrobenzoic acid; PDB, Protein Data Bank; TrxR, thioredoxin reductase; PAPS, phosphoadenosine phosphosulfate; Gdm-Cl, guanidinium chloride; DTNB, 5,5-dithiobis-(2-nitrobenzoic acid); hTrx, human Trx; ecTrx, E. coli Trx; IPTG, isopropyl -D-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride; S-MetO, S-methionine sulfoxide; MetO, methionine sulfoxide; LBCA, Last Bacterial Common Ancestor; LACA, Last Archaeal Common Ancestor; AECA, Archaeal–Eukaryotic Common Ancestor; LECA, Last Eukaryotic Common Ancestor; LAFCA, Last Animalia and Fungi Common Ancestor; LPBCA, Last Common Ancestor of the Cyanobacterial, Deinococcus, and Thermus group; LGPCA, Last ␥-Proteobacteria Common Ancestor. Further studies identified residues in the Trx fold with which the increased stabilities of ancestral thioredoxins correlate, allowing the prediction of stabilizing mutations [2, 19,20,21,22] Despite these important insights into the mechanisms underlying the evolution of thermostability in the thioredoxin fold, comparably little is known about the in vivo function of resurrected ancestral thioredoxins in the context of a modern mesophilic organism. The functional analysis of the ancestral thioredoxins in the reconstituted electron transport pathway from NADPH via TrxR, Trx, and MsrA to methionine sulfoxide in vitro and their in vivo function in the same electron transport chain under selective pressure indicated that thioredoxins and thioredoxin reductases co-evolved, and that the ability of thioredoxins to efficiently reduce multiple natural and non-natural substrates has been preserved throughout evolution
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