New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. We have used crystallography, NMR dynamics, kinetics and mass spectrometry to explain all of the molecular innovations that arose during a previous real-time evolution experiment. In this experiment, the (βα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bi-functional generalists. We discover that selection affected enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bi-functionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation; and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead we observed thresholds for each activity, above which further improvements in catalytic efficiency had little, if any, effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with activities that are sufficiently high that they no longer limit the growth of their host organism, and it confirms the (βα)8 barrel as an inherently evolvable protein scaffold.

Structural and functional innovations in the real-time evolution of new (βα)8 barrel enzymes

254th National Meeting (2017)

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