Ancestral versus Modern Substrate Scope in Family-1 Glycosidases

Promiscuity, the capability of catalyzing a diversity of chemical reactions, is a desirable feature in many enzymes intended for biotechnological applications. Promiscuous activities, however, are typically depressed in specialized modern enzymes. On the other hand, several ancestral reconstruction studies have reported enzymes with enhanced promiscuity. Glycosidases catalyze the hydrolysis of glycosidic bonds in all living cells but find practical applications in the synthesis of glycoconjugates as catalysts of the reverse reaction. Here, we use a library of about 500 possible substrates to compare the catalytic scope of the modern family-1 glycosidase from Halothermothrix orenii with that of a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for the hydrolysis of small substrates typically used to assess the activity of family-1 glycosidases. We identify, however, a trend for the modern enzyme to become less catalytically efficient with increasing substrate size. Such trend is not apparent in the conformationally-flexible ancestral glycosidase, which is in fact the better catalyst for the hydrolysis of flavonoid glycosides. Our results support that ancestral sequence reconstruction may provide a basis for enzyme engineering for the synthesis of glycoconjugates with large glycosyl acceptors.