Resurrected Ancestral TIM-Barrel Glycosidase Displays Heme Binding and Allosteric Modulation

Abstract

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. In this work, we report an ancestral sequence reconstruction exercise targeting ancient nodes during the evolution of family-1 glycosidases and present the exceptional properties of a putative resurrected ancestor of bacterial and eukaryotic enzymes. 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 for ∼1400 modern glycosidases show a porphyrin ring in the enzyme. 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 resurrection to reveal valuable but unusual protein features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.