Substrate scope of ancestral versus modern family-1 glycosidases.

Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related to the degradation and synthesis of carbohydrates and glycoconjugates. Herein, we explore how ancestral reconstruction impacts the substrate scope of glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the synthetic reaction. To this end, we screened 291 potential glycosidase substrates for degradation by both a modern family-1 glycosidase from the halothermophilic bacterium Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction of a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for many substrates, but the ancestral glycosidase is more efficient with flavonoid glycosides bearing large-aglycone moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, supports the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular modeling supports that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the protein and a cavity capable of accommodating large aglycones. Our results and analyses provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.