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 (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 | 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Bos taurus | Q9TTF5 | - |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
glycoprotein | N-linked glycosylation near the active site | Bos taurus |
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 | 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 | Comment | Organism |
---|---|---|
bCD38 | - |
Bos taurus |
CD38/NAD+ glycohydrolase | - |
Bos taurus |
NADase | - |
Bos taurus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Bos taurus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Bos taurus |
General Information | Comment | Organism |
---|---|---|
additional information | invariant glutamate 218 identified is the catalytic residue of the enzyme, Structure homology modelling, overview | Bos taurus |