Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks.Enzymes in bacteria and other organisms are built following instructions contained within each cell's DNA. Changes in the DNA, that is to say, mutations, can alter the shape and activity of the enzymes that are produced, which can ultimately affect the ability of the organism to survive and reproduce. Mutations that are beneficial to the organism are more likely to be passed on to future generations, which can lead to populations changing over time.The DNA sequences that an organism carries are referred to as its ‘genotype’ and the resulting physical characteristics of the organism are known as its ‘phenotype’. Studies of evolution tend to focus on how particular species or molecules become more different over time. However, one area that remains controversial is whether it is possible for evolution to be reversed so that an organism or molecule returns to a previous form.An enzyme called PTE is said to have phosphotriesterase activity because it catalyzes this particular type of chemical reaction. Recently, a group of researchers used a method called ‘directed evolution’ to demonstrate that it is possible for PTE to evolve in a way that means it loses its phosphotriesterase activity and becomes able to catalyze a different type of chemical reaction. Here, Kaltenbach et al.—including some of the researchers from the previous work—investigated whether it was possible to use the same method to reverse this evolution and restore the enzyme's original activity.The experiments show that reverse evolution is possible as phosphotriesterase activity was restored to the PTE enzyme from the previous study. However, although the phenotype of the final enzyme matched that of the original PTE enzyme, the genotypes did not match as the DNA sequences of the genes that encode these enzymes differ. The DNA does not revert to its original sequence because the effect of individual mutations on the phenotype depends on what other mutations are present. For example, as the enzyme evolved its new activity, additional mutations accumulated that did not alter enzyme activity. During the reverse evolution experiment, some of these mutations could have started to exert influence on the phenotype so that different mutations were required to restore the phosphotriesterase activity.In the future, Kaltenbach et al.'s findings may aid efforts to engineer artificial enzymes for use in medicine or industry.
Kaltenbach, M., Jackson, C. J., Campbell, E. C., Hollfelder, F., & Tokuriki, N. (2015). Reverse evolution leads to genotypic incompatibility despite functional and active site convergence. ELife, 4. https://doi.org/10.7554/elife.06492