Baier, FlorianHong, NansookYang, GloriaPabis, AnnaMiton, Charlotte M.Barrozo, AlexandreCarr, Paul DKamerlin, Shina CLJackson, ColinTokuriki, Nobuhiko2020-03-312020-03-31http://hdl.handle.net/1885/202526Genetic 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.script. Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN 418262–12 and RGPIN 2017–04909), and Canadian Institute of Health Research (CIHR) Foundation Grant to NT. NT is a CIHR new investigator and a Michael Smith Foundation of Health Research (MSFHR) career investigator. We also thank the Knut and Alice Wallenberg and Wenner-Gren Foundations for fellowships to SCLK and AP respectively, as well as the Swedish National Infrastructure for Computing (SNAC) for supercomputing resources.20 pagesapplication/pdfen-AU© Baier et al.https://creativecommons.org/licenses/by/4.0/2019-02-0510.7554/eLife.407892019-11-25This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.