Expanding the Substrate Specificity of Macro Domains toward 3″-Isomer of O-Acetyl-ADP-ribose

O-Acetyl-ADP-ribose (OAADPR) is a signaling molecule identified from the conserved sirtuin reaction inSaccharomyces cerevisiae, involved in the important cellular functions of gene silencing, redox regulation, and aging. Here, we performed biochemical and structural characterization of the yeast Poa1p macro domain in detail, uncovering an unusual deacetylase activity favoring 3?- and 1?-isomers ofO-acetyl-ADP-ribose. The unique active-site residues of Poa1p contributing to the distinct substrate specificity thus shed light on the divergent branch of a POA1-like subclass. Moreover, disruption of Poa1p expression in yeast showed a striking sensitivity to transcriptional stress, which implies a physiological role in response to nucleotide depletion. These findings provide biochemical and structural insights into a noncanonical 3?-O-acetyl-ADP-ribose deacetylase, which plays a critical role in cellular nucleotide metabolism for intracellular signaling and the regulatory process. ? 2021 American Chemical Society