Thermostable ancestors enabled evolutionary diversification of promiscuous chemical defence enzymes

Abstract Enzymes are generally believed to evolve from promiscuous ancestors to more specialized descendants under some selection pressure related to their function. However, enzymes whose function depends on substrate promiscuity have not been studied. Here, we show that a group of highly diverse, xenobiotic-metabolizing enzymes, responsible for defense against a constantly changing battery of xenobiotic chemicals, evolved from highly thermostable ancestors. Thermostability declined in parallel with the accumulation of sequence diversity through evolution. The major lineages differed in their relative diversification, with the more stable lineage leading to greater extant sequence diversity. Thermostability was associated with a trend towards better sequestration of hydrophobic residues within the core of the protein and increased exposure of polar residues in solvent-accessible parts of the structure. Resurrected ancestral forms were active towards typical substrates and exhibited ligand-binding promiscuity comparable to, or greater than, their extant descendants. This work supports the hypothesis that robust ancestors facilitate evolutionary diversification and highlights features responsible for enhancing thermostability in a protein fold.