Directed Evolution of a Model Primordial Enzyme Provides Insights into the Development of the Genetic Code

摘要

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function. Author Summary Proteins are linear polymers of a set of typically 20 different amino acid building blocks. The amino acid sequence—encoded by a genetic template—directs the folding of newly synthesized proteins into compact 3D structures and dictates the function of the protein product. Monomers containing distinct physico-chemical properties and geometries allow the formation of highly sophisticated architectures, and diverse functional groups enable enzymes to catalyze a plethora of chemical transformations. Nevertheless, the biochemical rationale for the exact composition (and particularly the redundancy) of the proteinogenic amino acid alphabet, which contains multiple building blocks that are chemically similar, remains enigmatic. By subjecting a simplified enzyme—constructed from only nine different amino acids—to directed evolution, we were able to investigate the impact of amino acid diversity on protein function. The most prolific variant selected in the course of the experiments expanded its amino acid alphabet, albeit through two surprisingly subtle mutations (isoleucine to threonine and leucine to valine). The mutations improve both stability and catalytic activity of the enzyme, thereby demonstrating that the presence of structurally similar amino acids specified by the genetic code is highly beneficial for protein fitness.