S155: Smooth and gradual transition to new enzyme function via bi-function and bi-conformational intermediates

Comprehending the process of protein evolution demands knowledge of the intermediates that underline evolutionary transitions between one function to another. We have evolved in the laboratory a new aryl esterase, starting from an existing phosphotriesterase that exhibits weak promiscuous aryl-esterae activity. Eighteen iterative cycles of random mutagenesis and selection led to 3x10⁴-fold activity gain to yield an efficient aryl esterase (k_cat/K_M ≈ 5x10⁶ s^-1 M^-1) and a loss of 10⁴-fold in the original activity. The mutation-by-mutation trajectory we followed was found to be ‘smooth’, i.e., evolution occurred by small steps through bi-functional intermediates. Structural and mechanistic analysis revealed how the active-site electrostatics and shape-complementarity were gradually altered by mutations within the active-site, but mostly allover the enzyme's scaffold. The gradual and smooth transitions were mediated by structural plasticity that enabled the intermediates to rearrange between different active site architectures, one conferring the original activity, and another, the newly evolving one.