Ancient Catalyst Uses Modern Tricks

Researchers have reconstructed an ancient thioredoxin enzyme and found that its strategy for reducing disulfide is far from primitive. The 4 billion-year-old enzyme catalyzes reduction the same way modern thioredoxins do, except that it works at lower pH, higher temperature, and with relaxed substrate specificity—characteristics useful for functioning in primordial Earth conditions and potentially in industrial applications (Nat. Struct. & Mol. Biol., DOI: 10.1038/nsmb.2020).

"We thought that the resurrected enzymes would use simple reducing chemistry, but shockingly, the very ancient enzymes worked like modern ones," says Julio Fernandez, the protein biologist at Columbia University who led the research.

Fernandez and his colleagues reconstructed the ancient enzyme by first tabulating hundreds of gene sequences of modern thioredoxin enzymes found in bacteria, animals, and plants. Then they used statistical algorithms to figure out a feasible sequence for a common evolutionary ancestor. Finally, they inserted that sequence into Escherichia coli bacteria and purified the resulting ancient enzyme.

The statistical algorithm suggested a primordial sequence that is 70% different from that of current thioredoxins. This major difference initially worried Fernandez, but not only did the protein fold into a three-dimensional structure similar to modern counterparts, it also reduced disulfide bonds.

Fernandez’ team used single-molecule force spectroscopy to figure out that the ancient thioredoxin binds substrate and reorients the substrate’s disulfide bond so an S_N2 reaction can take place—just like modern counterparts.

"This paper is certainly elegant," says Barry Hall, who recently retired from Rochester University, where he developed strategies for reconstructing ancient protein sequences." There are very likely practical engineering applications for paleobiochemistry," he adds. For example, industrial processes operate under conditions very similar to primordial Earth—high temperatures and sometimes low pH. If no extant enzymes function at that temperature, and if efforts to engineer or evolve mutant thermoactive enzymes fail, resurrecting enzymes that worked in primordial Earth could be a promising alternative, Hall notes.