Literature summary for 3.2.1.143 extracted from

Pourfarjam, Y.; Ventura, J.; Kurinov, I.; Cho, A.; Moss, J.; Kim, I.K.
Structure of human ADP-ribosyl-acceptor hydrolase 3 bound to ADP-ribose reveals a conformational switch that enables specific substrate recognition (2018), J. Biol. Chem., 293, 12350-12359 .

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Cloned(Commentary)

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 (Commentary)

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

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

Protein Variants	Comment	Organism
D314E	site-directed mutagenesis, poly(ADP-ribose) binding structures of wild-type and D314A mutant, overview	Homo sapiens

Metals/Ions

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

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9NX46	-	-

Purification (Commentary)

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

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

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	a

Substrates and Products (Substrate)

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

Synonyms	Comment	Organism
ADP-ribosyl-acceptor hydrolase 3	-	Homo sapiens
ADPRHL2	-	Homo sapiens
ARH3	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Homo sapiens

General Information

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