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.

C⁴‐substituted fluoroprolines (4R)‐fluoroproline ((4R)‐Flp) and (4S)‐fluoroproline ((4S)‐Flp) have been used in protein engineering to enhance the thermodynamic stability of peptides and proteins. The electron‐withdrawing effect of fluorine can bias the pucker of the pyrrolidine ring, influence the conformational preference of the preceding peptide bond, and can accelerate the cis/trans prolyl peptide bond isomerisation by diminishing its double bond character. The role of 4,4‐difluoroproline (Dfp) in the acceleration of the refolding rate of globular proteins bearing a proline (Pro) residue in the cis conformation in the native state remains elusive. Moreover, the impact of Dfp on the thermodynamic stability and bioactivity of globular proteins has been seldom described. In this study, we show that the incorporation of Dfp caused a redox state dependent and position dependent destabilisation of the thioredoxin (Trx) fold, while the catalytic activities of the modified proteins remained unchanged. The Pro to Dfp substitution at the conserved cisPro76 in the thioredoxin variant Trx1P did not elicited acceleration of the rate‐limiting trans‐to‐cis isomerization of the Ile75‐Pro76 peptide bond. Our results show that pucker preferences in the context of a tertiary structure could play a major role in protein folding, thus overtaking the rules determined for cis/trans isomerisation barriers determined in model peptides.

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2′-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi–Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.