Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of alpha(1-> 2') or alpha(1'-> 2) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to ail immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln(755)-(Glu-Glu) under bar (757), and a third residue found in the conserved sequence Val(737)-(Asp) under bar -Phe-Ala-Asn(741). Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr(796) and a glycine-rich region Gly(745)-Gly-Gly(747) important for PARG function, allowed us to define a PARG 'signature sequence' [vDFA-X-3-GGg-X6-8-vQEEIRF-X-3-PE-X-14-E-X-12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610-795 in bovine PARG.