Intramolecular Epistasis and the Evolution of a New Enzymatic Function
Figure 3
Step-wise laboratory-based evolution of AtzA to TriA.
Circles indicate the variants for which the kcat/KM values (s−1.M−1; values in Table 1 and Table S2) for atrazine dechlorination and melamine deamination were determined (color coded as follows: AtzA, red (filled); generation 1, orange (filled); generation 2, green (filled); generation 3, blue (filled); generation 4, violet (filled); generation 5, orange (open); generation 6, red (open); generation 7, green (open); generation 8, blue (open); and TriA, violet (open)). Lines are used to link variants differing by one substitution (thick lines link optimal variants; thin lines link the optimal variants to suboptimal variants – suboptimal variants were not used to generate subsequent variants). Amino acid substitutions discussed in the text have been labelled for clarity, as have the wild-type AtzA and TriA enzymes. Inset: expansion of the region of Fig. 3 that contains the last four steps of the trajectory.