Cloned (Comment) | Organism |
---|---|
expressed in Saccharomyces cerevisiae strain BJ5460 | Saccharomyces cerevisiae |
gene SCS7, recombinant overexpression of ZZ/His6/MBP-tagged wild-type scScs7p and DELTA95scScs7p from plasmid pSGP46 in Saccharomyces cerevisiae strain BJ5460, recombinant expression of point mutants in enzyme-deficient Saccharomyces cerevisiae strain | Saccharomyces cerevisiae |
Crystallization (Comment) | Organism |
---|---|
microbatch-under-oil method, using 20% polyethylene glycol 3350 (w/v), 100 mM HEPES, pH 7.0, 300 mM ammonium sulfate | Saccharomyces cerevisiae |
purified recombinant wild-type scScs7p and mutant DELTA95scScs7p, microbatch-under-oil method, micxing of 0.002 ml of protein solution at 2.5 or 3.3 mg/ml with 0.002 ml of crystallization solution containing 10% PEG 2000, 100 mM sodium citrate, pH 5.6, and 50 mM ammonium sulfate, or 20% w/v PEG 3350, 100 mM HEPES, pH 7.0, and 300 mM ammonium sulfate, and then covering the drop with 0.05 ml of mineral oil, 2-7 days, 14°C, soaking in heavy atom solution saturated with K2HgI4, X-ray diffraction structure determination and analysis at 2.6-6.0 A resolution | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
D323A | site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H173A | site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H244A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H249A | site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H264A | site-directed mutagenesis, the mutation results in unaltered syringomycin E sensitivity compared to the wild-type | Saccharomyces cerevisiae |
H268A | site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H271A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H272A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H326A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H330A | site-directed mutagenesis, the mutation results in complete loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H331A | site-directed mutagenesis, the mutation results in unaltered syringomycin E sensitivity compared to the wild-type | Saccharomyces cerevisiae |
H345A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H348A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
H349A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
additional information | generation of an scs7-DELTA strain from wild-type Saccharomyces cerevisiae strain BY4742. The model of DELTA95scScs7p mutant is inserted into a pre-equilibrated lipid bilayer comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine | Saccharomyces cerevisiae |
Y319A | site-directed mutagenesis, the mutation results in unaltered syringomycin E sensitivity compared to the wild-type | Saccharomyces cerevisiae |
Y322A | site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity | Saccharomyces cerevisiae |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
endoplasmic reticulum | - |
Saccharomyces cerevisiae | 5783 | - |
endoplasmic reticulum membrane | an integral membrane protein, the Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum | Saccharomyces cerevisiae | 5789 | - |
additional information | modeling of secondary structural elements of the scScs7p hydroxylase domain placed within a model lipid bilayer, overview | Saccharomyces cerevisiae | - |
- |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | the enzyme contains two zinc atoms | Saccharomyces cerevisiae | |
Zn2+ | the enzyme contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. The dimetal-binding site is located within the catalytic cap domain and is occupied by zinc atoms coordinated by the highly conserved histidine side chains from four histidine box motifs. Molecular dynamics simulations, allows modeling of a ceramide substrate below the dimetal center of scScs7p | Saccharomyces cerevisiae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
a phytoceramide + 2 ferrocytochrome b5 + O2 + 2 H+ | Saccharomyces cerevisiae | - |
a (2'R)-2'-hydroxyphytoceramide + 2 ferricytochrome b5 + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | Q03529 | - |
- |
Purification (Comment) | Organism |
---|---|
immunoglobulin G-Sepharose 6 resin column chromatography and Superdex 200 gel filtration | Saccharomyces cerevisiae |
recombinant tagged wild-type scScs7p and DELTA95scScs7p from Saccharomyces cerevisiae strain BJ5460 membranes by solubilization with beta-octyl glucoside, ultracentrifugation, affinity/immunoaffinity chromatography, and tag cleavage through rhinovirus 3C protease, which is removed by adding His-Select cobalt resin, followed by ultrafiltration and gel filtration | Saccharomyces cerevisiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
a phytoceramide + 2 ferrocytochrome b5 + O2 + 2 H+ | - |
Saccharomyces cerevisiae | a (2'R)-2'-hydroxyphytoceramide + 2 ferricytochrome b5 + H2O | - |
? | |
a phytoceramide + 2 ferrocytochrome b5 + O2 + 2 H+ | a phytoceramide, containing a 26-carbon very-long-chain fatty acid (VLCFA) moiety, is used as the model substrate | Saccharomyces cerevisiae | a (2'R)-2'-hydroxyphytoceramide + 2 ferricytochrome b5 + H2O | - |
? | |
additional information | molecular dynamics simulations, allows modeling of a ceramide substrate below the dimetal center of scScs7p | Saccharomyces cerevisiae | ? | - |
? | |
phytoceramide + ferrocytochrome b5 + O2 + H+ | - |
Saccharomyces cerevisiae | (2'R)-2'-hydroxyphytoceramide + ferricytochrome b5 + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the core structure is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The dimetal-binding site is located within the catalytic cap domain and is occupied by zinc atoms coordinated by the highly conserved histidine side chains from four histidine box motifs | Saccharomyces cerevisiae |
Synonyms | Comment | Organism |
---|---|---|
FA2H | - |
Saccharomyces cerevisiae |
fatty acid 2-hydroxylase | - |
Saccharomyces cerevisiae |
fatty acid alpha-hydroxylase | - |
Saccharomyces cerevisiae |
SCS7 | - |
Saccharomyces cerevisiae |
Scs7p | - |
Saccharomyces cerevisiae |
scScs7p | - |
Saccharomyces cerevisiae |
sphingolipid alpha-hydroxylase | - |
Saccharomyces cerevisiae |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
cytochrome b5 | the enzyme contains a cytochrome b5 domain fused to the catalytic domain at the N terminus of the enzyme | Saccharomyces cerevisiae |
General Information | Comment | Organism |
---|---|---|
evolution | FA2H/Scs7p belongs to a superfamily of integral membrane di-iron-containing enzymes that hydroxylate or desaturate lipid-based substrates via a reaction mechanism that is dependent on NADH and oxygen | Saccharomyces cerevisiae |
additional information | important role of the dimetal-binding histidines in catalysis, residues Tyr322 and Asp323 are critical determinants involved in the hydroxylase reaction. Molecular dynamics simulations, structure-function analysis, and generation of a model of ceramide binding to enzyme Scs7p | Saccharomyces cerevisiae |
physiological function | enzyme Scs7p is responsible for adding a hydroxyl group to the alpha-carbon of the VLCFA moiety of the ceramide substrate to generate inositol phosphoceramide, one of three major sphingolipids in yeast | Saccharomyces cerevisiae |