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Literature summary for 3.5.1.23 extracted from

  • Gebai, A.; Gorelik, A.; Li, Z.; Illes, K.; Nagar, B.
    Structural basis for the activation of acid ceramidase (2018), Nat. Commun., 9, 1621 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.5.1.23

Activating Compound

Activating Compound Comment Organism Structure
saposin-D a hydrophobic surface surrounding the substrate binding channel appears to be a site of membrane attachment where the enzyme accepts substrates facilitated by the accessory protein, saposin-D Homo sapiens

Cloned(Commentary)

Cloned (Comment) Organism
gene bwa, recombinant expression of His-tagged wild-type and mutant enzymes, the endogenous signal peptide comprising the first 21 residues is replaced by the melittin signal peptide (MKFLVNVALVFMVVYISYIYA) followed by a His6-tag. Constructs encompassed residues 22 to 395 of homologues from human (UniProt Q13510 variant Ile93Val), nmr (Heterocephalus glaber, UniProt A0A0P6JG37 variant Asn348Ser), or cmw (Balaenoptera acutorostrata scammoni, RefSeq XP_007174053). The latter two homologues are codon-optimized Homo sapiens

Protein Variants

Protein Variants Comment Organism
D331N naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
E138V naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
E180K naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
F136L naturally occuring disease mutation, F136 is located near the lipid tails of the modeled substrate and at the alpha-beta interface, the F136L mutation can destabilize the heterodimer and substrate interactions, affects the hydrophobic surface of the protein Homo sapiens
F328Q/F329Q/L330Q site-directed mutagenesis, hydrophobic patches mutated near the substrate binding channel, the mutant shows reduced ceramide hydrolysis compared with wild-type in the liposomal assay Homo sapiens
F87Q/V88Q/V93Q site-directed mutagenesis, mutation of a site further from the substrate-binding site, the mutant shows reduced ceramide hydrolysis compared with wild-type in the liposomal assay Homo sapiens
G168W naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
G235D naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
G235R naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
L182V naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
L80Q/V165Q/L167Q site-directed mutagenesis, hydrophobic patches mutated near the substrate binding channel, the mutant shows reduced ceramide hydrolysis compared with wild-type in the liposomal assay Homo sapiens
N320D naturally occuring disease mutation, active site residue mutation, disrupts the functional requirement for an asparagine side chain at this position Homo sapiens
N320D naturally occuring disease mutation, affects the activation of the proenzyme Homo sapiens
N320S naturally occuring disease mutation, active site residue mutation, disrupts the functional requirement for an asparagine side chain at this position Homo sapiens
N320S naturally occuring disease mutation, affects the activation of the proenzyme Homo sapiens
N320X naturally occuring disease mutation, active site residue mutation, inhibits autocleavage and/or substrate hydrolysis Homo sapiens
P362R naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
P362T naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
R226P naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
R254G naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
R333G naturally occuring disease mutation, active site residue mutation, that hinders the R333 function, affects the activation of the proenzyme Homo sapiens
R333H naturally occuring disease mutation, active site residue mutation, that hinders the R333 function, affects the activation of the proenzyme Homo sapiens
R333X naturally occuring disease mutation, active site residue mutation, inhibits autocleavage and/or substrate hydrolysis Homo sapiens
T179I naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
T222K naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
T42A naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
T42M naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
V97D naturally occuring disease mutation, the mutation inhibit the interaction of aCDase with negatively charged liposomes Homo sapiens
V97E naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens
V97G naturally occuring disease mutation, the mutation likely destabilizes helix-alpha2 in the alpha-subunit in which it resides Homo sapiens
W169Q/I171Q/W176Q site-directed mutagenesis, mutation of the L4-6 loop in the beta-subunit, the mutant shows reduced ceramide hydrolysis compared with wild-type in the liposomal assay Homo sapiens
W169R naturally occuring disease mutation, the mutation affects the hydrophobic surface of the protein Homo sapiens
Y36C naturally occuring disease mutation, the mutation is predicted to affect the folding or stability of the protein Homo sapiens

Localization

Localization Comment Organism GeneOntology No. Textmining
lysosome
-
 Homo sapiens 5764
-

Organism

Organism UniProt Comment Textmining
Homo sapiens Q9NUN7
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification the enzyme performs autocatalysis. In the proenzyme, the catalytic center is buried and protected from solvent. Autocleavage triggers a conformational change exposing a hydrophobic channel leading to the active site. Substrate modeling suggests distinct catalytic mechanisms for substrate hydrolysis versus autocleavage Homo sapiens

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes by nickel affinity chromatography and gel filtration Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ceramide + H2O ceramide in anionic liposomes. The negatively charged liposomes consisted of 25% mole bis(monoacylglycero)phosphate, 45% 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 20% cholesterol, and 10% 12:0 ceramide Homo sapiens ?
-
 ?
additional information the enzyme performs autocatalysis. Substrate modeling suggests distinct catalytic mechanisms for substrate hydrolysis versus autocleavage Homo sapiens ?
-
 ?

Subunits

Subunits Comment Organism
More hydrophobic surface of aCDase Homo sapiens

Synonyms

Synonyms Comment Organism
aCDase
-
 Homo sapiens
acid ceramidase
-
 Homo sapiens
ASAH1
-
 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
4
-
 assay at, substrate ceramide Homo sapiens
5
-
 assay at, autolytic cleavage Homo sapiens

General Information

General Information Comment Organism
malfunction abnormal function of the enzyme leads to Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, and is associated with Alzheimer's, diabetes, and cancer. Structural mapping of disease mutations reveals that most destabilize the protein fold Homo sapiens
additional information a hydrophobic surface surrounding the substrate binding channel appears to be a site of membrane attachment where the enzyme accepts substrates facilitated by the accessory protein, saposin-D. The shape of the substrate binding channel appears to be specific for ceramide, as other membrane-resident lipids with bulky head groups, such as sphingomyelin, phospholipids, and cerebrosides, result in steric clashes binding in a manner similar to the modelled ceramide. Catalytic mechanism of substrate hydrolysis, molecular docking and simulation, overview. Uncovering the substrate-binding site upon autocleavage, the concomitant conformational change to the alpha-beta junction causes strand-beta3 containing Cys 143 to shift. This moves Cys143 away from Arg159 breaking their putative hydrogen bond, suggesting that for substrate cleavage Arg159 may not act as the general base. Instead, the general base in the activated state is mostly likely to be the newly formed N-terminus of the beta-subunit. Active site residue N320 stabilizes the N-terminus of Cys143 through hydrogen bonding with its side chain oxygen, whereas its side chain nitrogen provides the oxyanion hole for substrate hydrolysis and stabilizes the position of Glu225, also important for oxyanion hole formation. R333 hydrogen bonds to N320 and based on the substrate modeling above, is predicted to be important for engaging ceramide Homo sapiens
physiological function acid ceramidase (aCDase, ASAH1) hydrolyzes lysosomal membrane ceramide into sphingosine, the backbone of all sphingolipids, to regulate many cellular processes Homo sapiens