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Literature summary for 3.2.2.6 extracted from

  • Egea, P.F.; Muller-Steffner, H.; Kuhn, I.; Cakir-Kiefer, C.; Oppenheimer, N.J.; Stroud, R.M.; Kellenberger, E.; Schuber, F.
    Insights into the mechanism of bovine CD38/NAD+ glycohydrolase from the X-ray structures of its Michaelis complex and covalently-trapped intermediates (2012), PLoS ONE, 7, e34918.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of bovine CD38/NAD+ glycohydrolase truncated for the first 31 amino acids that encompass the transmembrane and short intracellular domains in Pichia pastoris. The construct comprises a DNA fragment encoding the ecto-domain in the expression plasmid pPICZaA in frame with the yeast alpha-factor secretion signal sequence under the transcriptional control of the AOX1 promoter and keeping its original stop codon Bos taurus

Crystallization (Commentary)

Crystallization (Comment) Organism
mono N-glycosylated forms of soluble enzyme ecto-domain (residues 32-278) and catalytic residue mutant Glu218Gln, in apo state or bound to 2'-fluorinated NAD+ derivatives aFNAD or rFNAD, hanging drop vapour diffusion method, from 20-30% PEG 4000, 50-250 mM ammonium sulfate and 100 mM sodium cacodylate, sodium acetate or MES, pH 6.0-6.5, room temperature, soaking of crystals in 1-3 mM ligand solution, X-ray diffraction structure determination and analysis at 1.8 A resolution, molecular replacement Bos taurus

Protein Variants

Protein Variants Comment Organism
E218Q site-directed mutagenesis, catalytic site mutant, crystal structure analysis, almost inactive mutant Bos taurus
K120A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bos taurus
additional information construction of CD38/NAD+ glycohydrolase truncated for the first 31 amino acids that encompass the transmembrane and short intracellular domains Bos taurus
R216A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bos taurus
S185A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bos taurus
W168A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Bos taurus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady state kinetics Bos taurus
0.0171
-
NAD+ pH 7.4, 37°C, recombinant wild-type enzyme Bos taurus
0.0187
-
NAD+ pH 7.4, 37°C, recombinant mutant S185A Bos taurus
0.0229
-
NAD+ pH 7.4, 37°C, recombinant mutant R216A Bos taurus
0.0246
-
NAD+ pH 7.4, 37°C, recombinant mutant W168A Bos taurus
0.0247
-
NAD+ pH 7.4, 37°C, recombinant mutant E218Q Bos taurus
0.0277
-
NAD+ pH 7.4, 37°C, recombinant mutant K120A Bos taurus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Bos taurus the enzyme catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR) ?
-
?
NAD+ + H2O Bos taurus
-
ADP-D-ribose + nicotinamide
-
?

Organism

Organism UniProt Comment Textmining
Bos taurus Q9TTF5
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
glycoprotein N-linked glycosylation near the active site Bos taurus

Reaction

Reaction Comment Organism Reaction ID
NAD+ + H2O = ADP-D-ribose + nicotinamide + H+ structure-function analysis and reaction mechanism, overview. The nicotinamide-ribosyl bond of NAD+ is cleaved via a dissociative process with a late transition state, leading to a ribooxocarbenium ion reaction intermediate stabilized by the side-chain of invariant Glu218. This rate-determining step is followed by two nucleophilic reactions in competition: (i) an intermolecular pathway involving a rapid trapping from the b-face of this intermediate by a water molecule (NAD+ glycohydrolase activity) or by competing neutral nucleophiles such as pyridines (transglycosidation reactions) or alcohols (e.g., methanolysis), and (ii) an intramolecular reaction between N1 of the adenine ring and C19 (anomeric carbon) of the oxocarbenium ion leading to the formation of cyclic ADP-ribose (ADP-ribosyl cyclase activity). This latter reaction represents a kinetically minor step relative to solvolysis Bos taurus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the enzyme catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR) Bos taurus ?
-
?
NAD+ + H2O
-
Bos taurus ADP-D-ribose + nicotinamide
-
?

Synonyms

Synonyms Comment Organism
bCD38
-
Bos taurus
CD38/NAD+ glycohydrolase
-
Bos taurus
NADase
-
Bos taurus

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information Comment Organism
additional information invariant glutamate 218 identified is the catalytic residue of the enzyme, Structure homology modelling, overview Bos taurus