Literature summary for 1.14.18.6 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 .

Search for more literature for 1.14.18.6

Cloned(Commentary)

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 (Commentary)

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

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

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

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

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	Q03529	-	-

Purification (Commentary)

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

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

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

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