Abstract
Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.
Highlights
Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds
GH1 protein homologs are widely distributed in all three domains of life and representative sequences were collected from each domain, including characterized GH1 sequences obtained from Carbohydrate-active enzyme database (CAZy) as well as homologous sequences contained in GenBank
We revealed that ancestral levels of catalysis are substantially reduced with respect to those obtained for the modern glycosidase from Halothermothrix orenii, used here as comparison
Summary
Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. A new and important implication of sequence reconstruction is currently emerging linked to the realization that resurrected ancestral proteins may display properties that are desirable in scaffolds for enzyme engineering[4,5,6]. High stability and substrate/catalytic promiscuity have been described in a number of ancestral resurrection studies[5,7] These two features are known contributors to protein evolvability[8,9], which points to the potential of resurrected ancestral proteins as scaffolds for the engineering of new functionalities[4,10]. We find upon ancestral resurrection a diversity of unusual and unexpected biomolecular properties that suggest new engineering possibilities that go beyond the typical applications of protein family being characterized. The process typically follows a Koshland mechanism based on two catalytic carboxylic acid residues and, with very few exceptions, does not involve cofactors
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