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Literature summary extracted from

  • Kuhn, I.; Kellenberger, E.; Cakir-Kiefer, C.; Muller-Steffner, H.; Schuber, F.
    Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues (2014), Biochim. Biophys. Acta, 1844, 1317-1331.
    View publication on PubMed

Protein Variants

EC Number Protein Variants Comment Organism
3.2.2.6 D147A site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
3.2.2.6 E138A site-directed mutagenesis, the mutation causes a modest increase in the rate of NAD+ transformation which is proportional to its concentration. At 4.0 M, the rate increase is about 1.2fold and the formation of beta-1'-O-methyl ADP-ribose amounts to about 80% of the total reaction products. The observed selectivity in favor of methanolysis is similar to that of wild-type enzyme. The ADP-ribosyl cyclase activity of E138A mutant is more affected by the competing nucleophile, i.e. formation of ADP-ribose and cADPR are reduced by 75% and 90% respectively at 4.0 M methanol, the mutant shows an increase in ADP cyclization and higly reduced overall activity compared to the wild-type enzyme Bos taurus
3.2.2.6 E138Q site-directed mutagenesis, in the presence of methanol, mutant E138Q efficiently catalyzes the formation of beta-1'-O-methyl ADP-ribose. But in contrast with mutant E138A, and like the wild-type enzyme, solvolysis does not affect the overall turnover rate of NAD+ indicating that the formation of the E.ADP-ribosyl intermediate is still rate limiting Bos taurus
3.2.2.6 E218A site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
3.2.2.6 E218Q site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
3.2.2.6 W118A site-directed mutagenesis, the mutant shows a decrease of the catalytic rate compared to the wild-type enzyme Bos taurus
3.2.2.6 W118A/W181A site-directed mutagenesis, the mutant shows a decrease of the catalytic rate which is 16fold lower than the product of the effects of the two single mutations Bos taurus
3.2.2.6 W118F site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
3.2.2.6 W118H site-directed mutagenesis, the mutant shows a decrease in activity compared to the wild-type enzyme Bos taurus
3.2.2.6 W181A site-directed mutagenesis, the mutant shows a decrease of the catalytic rate and a reduced sensitivity to nicotinamide inhibition compared to the wild-type enzyme Bos taurus
3.2.2.6 W181F site-directed mutagenesis, the mutant shows a decrease in activity and an increase in ADP cyclization compared to the wild-type enzyme Bos taurus

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.2.2.6 1,N6-etheno NAD+
-
 Bos taurus
3.2.2.6 2'-deoxy-2'-beta-D-fluoroarabinofuranoside NAD+
-
 Bos taurus
3.2.2.6 2'-deoxy-2'-beta-D-fluororibofuranoside NAD+ non-covalent complex of the inhibitor formed with enzyme mutant E218Q, PDB ID: 3ghh, and with wild-type enzyme, PDB ID: 3kou Bos taurus
3.2.2.6 3-aminopyridine
-
 Bos taurus
3.2.2.6 Isonicotinic acid hydrazide
-
 Bos taurus
3.2.2.6 nicotinamide inhibition involves enzyme residue Trp181 Bos taurus
3.2.2.6 Pyridine non-competitive inhibition of mutant E218A by pyridine Bos taurus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.2.6 0.0171
-
 NAD+ recombinant wild-type enzyme, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0175
-
 NAD+ recombinant mutant D147A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0189
-
 NAD+ recombinant mutant W118A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0215
-
 NAD+ recombinant mutant W181F, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0247
-
 NAD+ recombinant mutant E218Q, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0276
-
 NAD+ recombinant mutant E138A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.028
-
 NAD+ recombinant mutant E218A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0289
-
 NAD+ recombinant mutant W118H, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0295
-
 NAD+ recombinant mutant E138Q, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0297
-
 NAD+ recombinant mutant W118A/W181A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.036
-
 NAD+ recombinant mutant W118F, pH 7.4, 37°C Bos taurus
3.2.2.6 0.0453
-
 NAD+ recombinant mutant W181A, pH 7.4, 37°C Bos taurus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.2.6 NAD+ + H2O Bos taurus bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism ADP-D-ribose + nicotinamide
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.2.2.6 Bos taurus Q9TTF5
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
3.2.2.6 NAD+ + H2O = ADP-D-ribose + nicotinamide + H+ substrate binding with a crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD+, stepwise ordered uni-bi kinetic mechanism, overview. Residues Trp118, Glu138, Asp147, Trp181 stabilize the ribooxocarbenium ion-like transition state mostly by electrostatic interactions Bos taurus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.2.6 additional information selectivity in favor of methanolysis by wild-type enzyme and mutant E138A. 1'-Azido ADP-ribose is the reaction product obtained in the presence of azide. the ADP-ribosyl cyclase activity of wild-type bCD38 isminimal Bos taurus ?
-
 ?
3.2.2.6 NAD+ + H2O bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism Bos taurus ADP-D-ribose + nicotinamide
-
 ?
3.2.2.6 NAD+ + H2O via a stepwise reaction mechanism Bos taurus ADP-D-ribose + nicotinamide
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.2.6 bCD38
-
 Bos taurus
3.2.2.6 CD38/NAD+ glycohydrolase
-
 Bos taurus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.2.6 37
-
 assay at Bos taurus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.2.6 0.0155
-
 NAD+ recombinant mutant E218A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.016
-
 NAD+ recombinant mutant W118A/W181A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.03
-
 NAD+ recombinant mutant E218Q, pH 7.4, 37°C Bos taurus
3.2.2.6 0.122
-
 NAD+ recombinant mutant W118A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.49
-
 NAD+ recombinant mutant W181A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.81
-
 NAD+ recombinant mutant E138A, pH 7.4, 37°C Bos taurus
3.2.2.6 3.5
-
 NAD+ recombinant mutant D147A, pH 7.4, 37°C Bos taurus
3.2.2.6 6.64
-
 NAD+ recombinant mutant E138Q, pH 7.4, 37°C Bos taurus
3.2.2.6 7.8
-
 NAD+ recombinant mutant W181F, pH 7.4, 37°C Bos taurus
3.2.2.6 10.36
-
 NAD+ recombinant mutant W118F, pH 7.4, 37°C Bos taurus
3.2.2.6 18.37
-
 NAD+ recombinant mutant W118H, pH 7.4, 37°C Bos taurus
3.2.2.6 57.9
-
 NAD+ recombinant wild-type enzyme, pH 7.4, 37°C Bos taurus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.2.6 7.4
-
 assay at Bos taurus

General Information

EC Number General Information Comment Organism
3.2.2.6 additional information structure-function analysis, overview. The enzyme catalyzes the formation of beta-1'-O-methyl ADP-ribose in presence of methanol, solvolysis does not affect the overall turnover rate of NAD+ by the wild-type enzyme. Precise role of key conserved active site residues Trp118, Glu138, Asp147, Trp181 and Glu218, effects of experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. Binding of 2'-fluorinated analogs of NAD+ and trappping of the reaction intermediate, detailed overview. Catalytic residue Glu138 is part of the TLEDTL signature domain, Asp147 is a highly conserved residue in the enzyme and is important for the catalytic parameters. Cooperative contribution of Trp118 and Trp181 to catalysis Bos taurus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.2.6 0.54
-
 NAD+ recombinant mutant W118A/W181A, pH 7.4, 37°C Bos taurus
3.2.2.6 0.55
-
 NAD+ recombinant mutant E218A, pH 7.4, 37°C Bos taurus
3.2.2.6 1.21
-
 NAD+ recombinant mutant E218Q, pH 7.4, 37°C Bos taurus
3.2.2.6 6.4
-
 NAD+ recombinant mutant W118A, pH 7.4, 37°C Bos taurus
3.2.2.6 10.8
-
 NAD+ recombinant mutant W181A, pH 7.4, 37°C Bos taurus
3.2.2.6 29.3
-
 NAD+ recombinant mutant E138A, pH 7.4, 37°C Bos taurus
3.2.2.6 200
-
 NAD+ recombinant mutant D147A, pH 7.4, 37°C Bos taurus
3.2.2.6 220
-
 NAD+ recombinant mutant E138Q, pH 7.4, 37°C Bos taurus
3.2.2.6 290
-
 NAD+ recombinant mutant W118F, pH 7.4, 37°C Bos taurus
3.2.2.6 360
-
 NAD+ recombinant mutant W181F, pH 7.4, 37°C Bos taurus
3.2.2.6 630
-
 NAD+ recombinant mutant W118H, pH 7.4, 37°C Bos taurus
3.2.2.6 3380
-
 NAD+ recombinant wild-type enzyme, pH 7.4, 37°C Bos taurus