Literature summary for 3.5.1.23 extracted from

Vasiliauskaite-Brooks, I.; Healey, R.D.; Rochaix, P.; Saint-Paul, J.; Sounier, R.; Grison, C.; Waltrich-Augusto, T.; Fortier, M.; Hoh, F.; Saied, E.M.; Arenz, C.; Basu, S.; Leyrat, C.; Granier, S.
Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy (2018), Nat. Commun., 9, 5437 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene bwa, recombinant expression of wild-type and mutant enzymes in Spodoptera frugiperda Sf9 cells via baculovirus transfection system, the enzyme is cloned with C-terminal BRIL soluble module (thermostabilized apocytochrome b562RIL) and an N-terminal Flag epitope followed by a tobacco etch virus protease site	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type and mutant BRIL-fusion ACER3 enzyme reconstituted into 10:1 monoolein:cholesterol at a ratio of 1:1.5 protein:lipid by weight, from precipitant solution consisting of 34-40% PEG 400, 0.1 M HEPES, pH 7.5, 75 mM magnesium sulfate, and 5% DMSO, 5 days, X-ray diffraction structure determination and analysis at 2.70-2.85 A resolution	Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D19G	site-directed mutagenesis, partial loss of catalytic activity	Homo sapiens
E22G	site-directed mutagenesis, high loss of catalytic activity	Homo sapiens
E33G	naturally occuring inactive mutant, the mutation results in the destabilization of the calcium binding site	Homo sapiens
N24G	site-directed mutagenesis, high loss of catalytic activity	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
membrane	ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture	Homo sapiens	16020	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Ca2+	ACER3 has a catalytic Zn2+ binding site in its core and a Ca2+ binding site physically and functionally connected to the Zn2+, two residues appear to play critical roles in physically linking the Ca2+ and the Zn2+ sites, W20 and E33. E33 carboxylate interacts with the Ca2+ ion and is in close proximity to H81TM2 side chain, while H81TM2 coordinates the Zn2+, Ca2+-binding site within the N-terminus, structure overview. The Ca2+ ion is coordinated by six oxygens from the D19 carboxylate (bidentate), the W20 backbone carbonyl, the E22 backbone carbonyl, the N24 side chain carbonyl, and the E33 carboxylate (monodentate)	Homo sapiens
Zn2+	dependent on, ACER3 has a catalytic Zn2+ binding site in its core and a Ca2+ binding site physically and functionally connected to the Zn2+. Two residues appear to play critical roles in physically linking the Ca2+ and the Zn2+ sites, W20 and E33. E33 carboxylate interacts with the Ca2+ ion and is in close proximity to H81TM2 side chain, while H81TM2 coordinates the Zn2+. Zn2+ is coordinated by three His residues (H81TM2, H217TM7, and H221TM7) and a water molecule forming hydrogen bonds with S77TM2 and D92TM3. The residues forming this Zn2+ binding site are strictly conserved among the ACER family, and across species from yeast to human	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9NUN7	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant BRIL-fusion FLAG-tagged wild-type and mutant enzymes from Spodoptera frugiperda Sf9 cells by immunoaffinity and FLAG-tag affinity chromatography, the FLAG-tag is cleaved off by TEV protease, followed by gel filtration. The purified E33G ACER3 mutant is highly unstable during purification	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
N-oleoyl-D-sphingosine + H2O	-	Homo sapiens	oleate + sphingosine	-	?

Synonyms

Synonyms	Comment	Organism
ACER type 3	-	Homo sapiens
ACER3	-	Homo sapiens
alkaline ceramidase 3	-	Homo sapiens
intramembrane ceramidase	-	Homo sapiens

General Information

General Information	Comment	Organism
evolution	the fold of ACER3 is similar to adiponectin receptors (ADIPORs), structure comparisons, overview	Homo sapiens
malfunction	loss of function in E33G-ACER3 mutant found in leukodystrophic patients, ACER3 deficiency leads to progressive leukodystrophy in early childhood, mutation impaires the ACER3 ceramidases activity in patients' cells. This loss of function results in higher level of several ceramide species in the blood, in particular for the ACER3 preferred substrates, C18:1 and C20:1 ceramides. It is proposed that these aberrant levels of ceramides in the brain result in an incorrect central myelination leading to the clinical phenotype associated with the ACER3 mutant, i.e., neurological regression at 6-13 months of age, truncal hypotonia, appendicular spasticity, dystonia, optic disc pallor, peripheral neuropathy, and neurogenic bladder	Homo sapiens
additional information	substrate docking and hydrolysis mechanism, molecular dynamics simulation and docking study, overview. Computational docking of N-oleoyl-D-sphingosine	Homo sapiens
physiological function	alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. ACER3 has a catalytic Zn2+ binding site in its core and a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity	Homo sapiens

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Release 2023.1 (January 2023)