Cloned (Comment) | Organism |
---|---|
gene ADPRHL2, recombinant expression of His-tagged human PARG catalytic domain (residues 448-976) from Escherichia coli strain Rosetta (DE3), recombinant expression of His-tagged full-length wild-type and mutant D314E enzymes in Escherichia coli strain BL21(DE3) | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
purified recombinant full-length ARH3 wild-type enzyme and D314E mutant in complex with ADP-ribose and Mg2+, hanging drop vapor diffusion method, 10 mg/ml protein in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, and 5% glycerol, is mixed with reservoir solution containing 22% PEG 4000, 0.1 M sodium acetate, pH 4.5, and 0.1 M MgSO4, at 22°C, crystals are briefly equilibrated in a harvesting solution containing 26% PEG 4000, 0.1 M sodium acetate, pH 4.5, 0.1 M MgSO4, and 5 mM ADP-ribose, transferred to a cryoprotectant solution (26% PEG 4000, 0.1 M sodium acetate, pH 4.5, 0.1 M MgSO4, 5 mM ADPR, and 15% glycerol), and then flash-cooled in liquid nitrogen for data collection, X-ray diffraction structure determination and analysis at 1.6-1.7 A resolution, molecular replacement using the apo-ARH3DELTAN16 structure as a search model | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
D314E | site-directed mutagenesis, poly(ADP-ribose) binding structures of wild-type and D314A mutant, overview | Homo sapiens |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required for activity, two MG2+ with catalytic function, mechanism, overview. Asp314 is essential for the formation of the binuclear metal center | Homo sapiens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | Q9NX46 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant human PARG catalytic domain (residues 448-976) from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography, heparin chromatography, and gel filtration, recombinant His-tagged full-length wild-type and mutant D314E enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage through PreScission protease, and gel filtration, followed by ultrafiltration | Homo sapiens |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(ADP-ribose)n + H2O = (ADP-ribose)n-1 + ADP-ribose | Asp314 is located proximal to the 1''-O-linkage in substrates. Asp314 might protonate the leaving group (general acid), forming an oxocarbenium ion intermediate, and then activate the water (general base) for back-side attack. The W1 ligand of MgB can serve as the nucleophile attacking the anomeric C1'' of the ribose'' | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | assay using the PARylated PARP1 substrates, poly(ADP-ribose)-linked N-terminal DNA-binding domain of PARP1, i.e. PAR polymerase 1, a nicked DNA. Poly(ADP-ribose) binding structures of wild-type and D314A mutant, overview | Homo sapiens | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
ADP-ribosyl-acceptor hydrolase 3 | - |
Homo sapiens |
ADPRHL2 | - |
Homo sapiens |
ARH3 | - |
Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Homo sapiens |
General Information | Comment | Organism |
---|---|---|
evolution | full-length ARH3 (ARH3FL) adopts a compact all-alpha-helical fold with a central deep ADPR-binding cleft, a signature of the ARH3 superfamily | Homo sapiens |
additional information | proposed catalytic role of residue Asp314. Asp314 is located proximal to the 1''-O-linkage in substrates. Asp314 might protonate the leaving group (general acid), forming an oxocarbenium ion intermediate, and then activate the water (general base) for back-side attack. The W1 ligand of MgB can serve as the nucleophile attacking the anomeric C1'' of the ribose''. This is consistent with the observed O18 incorporation during hydrolysis of O-acetyl-ADP-ribose, reaction mechanism, overview. Asp314 is essential for the formation of the binuclear metal center. A conformational switch of ARH3 enables specific substrate recognition. ARH3 specifically exposes the scissile 1''-O-linkage in substrates for cleavage | Homo sapiens |
physiological function | enzyme ARH3 is a multifunctional enzyme that also hydrolyzes poly(ADP-ribose) (PAR). ARH3 can specifically hydrolyze PAR, mono-ADP-ribose post-translational modifications (MARPTMs), and O-acetyl-ADP-ribose. For all these substrates, ARH3 preferentially hydrolyzes the scissile alpha-O-linkage attached to the anomeric C1'' position of ADPR. In mammals, two enzymes, ADP-ribosyl-acceptor hydrolase 3 (ARH3 or ADPRHL2) and PAR glycohydrolase (PARG), function in tandem to reverse PARylation. These hydrolytic enzymes commonly cleave the alpha(1''-2') O-glycosidic linkages in PAR chains. ARH3 appears to catalyze primarily exocytic cleavage of PAR, generating free ADPR. It is reported that ARH3 protects cells from oxidative stress-induced parthanatos by lowering the cytoplasmic PAR level. ARH3 is a distinctive, multitasking enzyme that controls two biologically important NAD+-dependent cellular signaling pathways | Homo sapiens |