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 an 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 Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG 'signature sequence' [vDFA-X3-GGg-X6-8-vQEEIRF-X3-PE-X14-E-X12-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.
Patel
Chandra N
CN
Department of Pharmaceutical Sciences, College of Pharmacy, Markey Cancer Center and Center for Structural Biology, University of Kentucky, Lexington, KY 40536, USA.
Koh
David W
DW
Jacobson
Myron K
MK
Oliveira
Marcos A
MA
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase