Literature summary for 3.5.1.23 extracted from

Airola, M.V.; Allen, W.J.; Pulkoski-Gross, M.J.; Obeid, L.M.; Rizzo, R.C.; Hannun, Y.A.
Structural basis for ceramide recognition and hydrolysis by human neutral ceramidase (2015), Structure, 23, 1482-1491 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant overexpression of C-terminally His6-tagged extracellular region of human nCDase (residues 99-780) lacking the flexible O-glycosylated mucin box in Spodoptera frugiperda Sf9 cells, the enzyme is secreted	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His6-tagged extracellular domain of nCDase, residues 99-780, in complex with phosphate, hanging drop vapor diffusion, mixing of equal volumes of 3 mg/ml protein in 100 mM NaCl, 10 mM HEPES, pH 7.0, with reservoir solution containing 0.2 M Li2SO4, 10% PEG 1000, and 0.1 M citrate-phosphate, pH 4.6, at 16°C, X-ray diffraction structure determination and analysis at 2.6 A resolution, molecular replacement using Pseudomonas aeruginosa bCDase structure, PDB ID 2ZWS, as template	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant His6-tagged extracellular domain of nCDase, residues 99-780, in complex with phosphate, hanging drop vapor diffusion, mixing of equal volumes of 3 mg/ml protein in 100 mM NaCl, 10 mM HEPES, pH 7.0, with reservoir solution containing 0.2 M Li2SO4, 10% PEG 1000, and 0.1 M citrate-phosphate, pH 4.6, at 16°C, X-ray diffraction structure determination and analysis at 2.6 A resolution, molecular replacement using Pseudomonas aeruginosa bCDase structure, PDB ID 2ZWS, as template

Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.033	-	NBD-C12-ceramide	pH 7.0, 28°C, recombinant enzyme	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	required	Homo sapiens
Zn2+	dependent on	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9NR71	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant secreted His6-tagged nCDase (residues 99-780) from Spodoptera frugiperda Sf9 cells by nickel affinity chromatography and gel filtration	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
NBD-C12-ceramide + H2O	-	Homo sapiens	?	-	?

Synonyms

Synonyms	Comment	Organism
nCDase	-	Homo sapiens
neutral ceramidase	-	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.7	-	NBD-C12-ceramide	pH 7.0, 28°C, recombinant enzyme	Homo sapiens

General Information

General Information	Comment	Organism
additional information	the human enzyme contains a 20 A deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases. Flexible ligand docking and prediction of a binding mode for ceramide. The nCDase uses a distinct catalytic strategy for Zn2+-dependent amidases, and generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl headgroup of ceramide. Docking study with ligand C16-ceramide	Homo sapiens
physiological function	neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate. Enzyme nCDase regulates the balance of ceramide and sphingosine-1-phosphate	Homo sapiens

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
21.21	-	NBD-C12-ceramide	pH 7.0, 28°C, recombinant enzyme	Homo sapiens