Abstract
Genetic variation among orthologous proteins can cause cryptic phenotypic properties that only manifest in changing environments. Such variation may impact the evolvability of proteins, but the underlying molecular basis remains unclear. Here, we performed comparative directed evolution of four orthologous metallo-β-lactamases toward a new function and found that different starting genotypes evolved to distinct evolutionary outcomes. Despite a low initial fitness, one ortholog reached a significantly higher fitness plateau than its counterparts, 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 how cryptic molecular properties and conformational variation of active site residues in the initial genotypes cause epistasis, that could lead to distinct evolutionary outcomes. Our work highlights the importance of understanding the molecular details that connect genetic variation to protein function to improve the prediction of protein evolution.
Highlights
Genetic diversity across orthologous proteins is thought to be predominantly neutral with respect to their native, physiological function, but can cause variation in other non-physiological phenotypic properties, a phenomenon known as ‘cryptic genetic variation’ (Le Rouzic and Carlborg, 2008; Gibson and Dworkin, 2004; Paaby and Rockman, 2014)
Recent studies have shown that intramolecular epistasis is prevalent and that the phenotypic effect of the same mutation can be drastically altered when introduced into a genotype that differs by only a few other mutations, which may lead to different evolutionary outcomes depending on the starting genotype
The catalytic efficiencies for the native b-lactams hydrolysis are similar across these enzymes, their kcat/KM for the promiscuous phosphonate monoester hydrolase (PMH) activity varies by ~20 fold (Figure 1A and Supplementary file 1B)
Summary
Genetic diversity across orthologous proteins is thought to be predominantly neutral with respect to their native, physiological function, but can cause variation in other non-physiological phenotypic properties, a phenomenon known as ‘cryptic genetic variation’ (Le Rouzic and Carlborg, 2008; Gibson and Dworkin, 2004; Paaby and Rockman, 2014). Cryptic genetic variation has been shown to play an important role in evolution because genetically diverse populations are more likely to contain genotypes with a ‘pre-adapted’ phenotype, for example, a latent promiscuous function, that may confer an immediate selective advantage when the environment changes and a new selection pressure emerges (Queitsch et al, 2002; Specchia et al, 2010; de Visser et al, 2011; Amitai et al, 2007; Bloom et al, 2007; Rohner et al, 2013). The degree to which a trait can improve and the level of fitness that it can reach determines the evolutionary potential and outcome of a given genotype (Romero and Arnold, 2009).
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