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Literature summary extracted from

  • Zhu, G.; Koszelak-Rosenblum, M.; Connelly, S.M.; Dumont, M.E.; Malkowski, M.G.
    The crystal structure of an integral membrane fatty acid alpha-hydroxylase (2015), J. Biol. Chem., 290, 29820-29833 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.18.6 expressed in Saccharomyces cerevisiae strain BJ5460 Saccharomyces cerevisiae
1.14.18.6 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 (Commentary)

EC Number Crystallization (Comment) Organism
1.14.18.6 microbatch-under-oil method, using 20% polyethylene glycol 3350 (w/v), 100 mM HEPES, pH 7.0, 300 mM ammonium sulfate Saccharomyces cerevisiae
1.14.18.6 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

EC Number Protein Variants Comment Organism
1.14.18.6 D323A site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H173A site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H244A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H249A site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H264A site-directed mutagenesis, the mutation results in unaltered syringomycin E sensitivity compared to the wild-type Saccharomyces cerevisiae
1.14.18.6 H268A site-directed mutagenesis, the mutation results in partial loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H271A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H272A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H326A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H330A site-directed mutagenesis, the mutation results in complete loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H331A site-directed mutagenesis, the mutation results in unaltered syringomycin E sensitivity compared to the wild-type Saccharomyces cerevisiae
1.14.18.6 H345A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H348A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 H349A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae
1.14.18.6 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
1.14.18.6 Y319A site-directed mutagenesis, the mutation results in unaltered syringomycin E sensitivity compared to the wild-type Saccharomyces cerevisiae
1.14.18.6 Y322A site-directed mutagenesis, the mutation results in major loss of syringomycin E sensitivity Saccharomyces cerevisiae

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.14.18.6 endoplasmic reticulum
-
 Saccharomyces cerevisiae 5783
-
1.14.18.6 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
-
1.14.18.6 additional information modeling of secondary structural elements of the scScs7p hydroxylase domain placed within a model lipid bilayer, overview Saccharomyces cerevisiae
-
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.18.6 Zn2+ the enzyme contains two zinc atoms Saccharomyces cerevisiae
1.14.18.6 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/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.18.6 a phytoceramide + 2 ferrocytochrome b5 + O2 + 2 H+ Saccharomyces cerevisiae
-
 a (2'R)-2'-hydroxyphytoceramide + 2 ferricytochrome b5 + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.18.6 Saccharomyces cerevisiae Q03529
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.18.6 immunoglobulin G-Sepharose 6 resin column chromatography and Superdex 200 gel filtration Saccharomyces cerevisiae
1.14.18.6 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.18.6 a phytoceramide + 2 ferrocytochrome b5 + O2 + 2 H+
-
 Saccharomyces cerevisiae a (2'R)-2'-hydroxyphytoceramide + 2 ferricytochrome b5 + H2O
-
 ?
1.14.18.6 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
-
 ?
1.14.18.6 additional information molecular dynamics simulations, allows modeling of a ceramide substrate below the dimetal center of scScs7p Saccharomyces cerevisiae ?
-
 ?
1.14.18.6 phytoceramide + ferrocytochrome b5 + O2 + H+
-
 Saccharomyces cerevisiae (2'R)-2'-hydroxyphytoceramide + ferricytochrome b5 + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.18.6 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

EC Number Synonyms Comment Organism
1.14.18.6 FA2H
-
 Saccharomyces cerevisiae
1.14.18.6 fatty acid 2-hydroxylase
-
 Saccharomyces cerevisiae
1.14.18.6 fatty acid alpha-hydroxylase
-
 Saccharomyces cerevisiae
1.14.18.6 SCS7
-
 Saccharomyces cerevisiae
1.14.18.6 Scs7p
-
 Saccharomyces cerevisiae
1.14.18.6 scScs7p
-
 Saccharomyces cerevisiae
1.14.18.6 sphingolipid alpha-hydroxylase
-
 Saccharomyces cerevisiae

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.18.6 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

EC Number General Information Comment Organism
1.14.18.6 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
1.14.18.6 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
1.14.18.6 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