Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may also impact the evolutionary potential of proteins, but the molecular basis for this remains unclear. Here we perform comparative directed evolution in which four orthologous metallo-β- lactamases were evolved toward a new function. We found that genetic variation between these enzymes resulted in distinct evolutionary outcomes. The ortholog with the lower initial activity reached a 20-fold higher fitness plateauexclusively via increasing catalytic activity. By contrast, the ortholog with the highest initial activity evolved to a less-optimal and phenotypically distinct outcome through changes in expression, oligomerization and activity. We show that the cryptic molecular properties and conformational variation of residues in the initial genotypes cause epistasis, thereby constraining evolutionary outcomes. Our work highlights that understanding the molecular details relating genetic variation to protein functions is essential to predicting the evolution of proteins.

Cryptic genetic variation defines the adaptive evolutionary potential of enzymes