Heme-binding enables allosteric modulation in an ancient TIM-barrel glycosidase

Abstract Glycosidases are phylogenetically widely distributed enzymes that crucial for the cleavage of glycosidic bonds. Here, we present exceptional properties a putative ancestor bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares TIM-barrel fold with its modern descendants but displays large regions greatly enhanced conformational flexibility. Yet, barrel core remains comparatively rigid glycosidase activity is stable, an optimum temperature within experimental range thermophilic None ?5500 reported crystallographic structures ?1400 glycosidases show bound porphyrin. Remarkably, binds heme tightly stoichiometrically at well-defined buried site. Heme binding rigidifies this allosterically enhances catalysis. Our work demonstrates capability reconstructions to reveal valuable unexpected biomolecular features when sampling distant sequence space. potential as scaffold custom catalysis biosensor engineering discussed.