Literature summary for 2.7.1.91 extracted from

Wang, Z.; Min, X.; Xiao, S.H.; Johnstone, S.; Romanow, W.; Meininger, D.; Xu, H.; Liu, J.; Dai, J.; An, S.; Thibault, S.; Walker, N.
Molecular basis of sphingosine kinase 1 substrate recognition and catalysis (2013), Structure, 21, 798-809.

Application

Application	Comment	Organism
drug development	the enzyme is a potential target for drug discovery	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in High Five cells using the baculovirus transfection method	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant truncated enzyme comprising residues 9-364, in apo-form and in complex with inhibitor SKI-II, and with inhibitor SKI-II and ADP, sitting drop vapour diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 50 mM Tris-HCl, pH 8.5, 300 mM NaCl, 0.5 M GdnHCl, 10% glycerol, 0.1% Tween 20, and 10 mM DTT, and for complex crystals with a 3fold excess of inhibitor, and ATP/ADP, with 0.001 ml of well solution containing 1.0-1.4 M ammonium sulfate, 0.3-1.3 M NaCl, and 0.1 M Bis-Tris, pH 6.0, 16°C, X-ray diffraction structure determination and analysis at 2.0-2.3 A resolution, molecular replacement	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant truncated enzyme comprising residues 9-364, in apo-form and in complex with inhibitor SKI-II, and with inhibitor SKI-II and ADP, sitting drop vapour diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 50 mM Tris-HCl, pH 8.5, 300 mM NaCl, 0.5 M GdnHCl, 10% glycerol, 0.1% Tween 20, and 10 mM DTT, and for complex crystals with a 3fold excess of inhibitor, and ATP/ADP, with 0.001 ml of well solution containing 1.0-1.4 M ammonium sulfate, 0.3-1.3 M NaCl, and 0.1 M Bis-Tris, pH 6.0, 16°C, X-ray diffraction structure determination and analysis at 2.0-2.3 A resolution, molecular replacement

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D81A	site-directed mutagenesis, the mutant shows essentially no activity for phosphoryl transfer and ADP formation	Homo sapiens
D81N	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Homo sapiens
additional information	construction of an N- and C-terminally truncated enzyme form comprising residues 9-364 of full-length 368 residues, the mutant is catalytically active	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
SKI-II	highly selective and non ATP-competitive, enzyme binding structure, overview	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics	Homo sapiens
0.02	-	ATP	recombinant wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.0255	-	ATP	recombinant mutant D81N enzyme, pH and temperature not specified in the publication	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, binding structure, overview	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + sphingosine	Homo sapiens	-	ADP + sphingosine 1-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9NYA1	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged wild-type and mutant enzymes from High Five cells by nickel affinity chromatography, the tag is cleaved by TEV protease, followed by anion and cation exchange chromatographies, and gel filtration	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + D-erythro-sphingosine	-	Homo sapiens	ADP + D-erythro-sphingosine 1-phosphate	nucleotide binding in the cleft between the N-terminal domain and C-terminal domain	?
ATP + sphingosine	-	Homo sapiens	ADP + sphingosine 1-phosphate	-	?

Subunits

Subunits	Comment	Organism
More	the SphK1 structure reveals a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain, structure comparisons with Staphylococcus aureus DgkB, PDB ID 2QVL, and Archaeoglobus fulgidus NAD kinase, PDB ID 1ZOZ	Homo sapiens

Synonyms

Synonyms	Comment	Organism
sphingosine kinase 1	-	Homo sapiens
SPHK1	-	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.0065	-	ATP	recombinant mutant D81N enzyme, pH and temperature not specified in the publication	Homo sapiens
0.0427	-	ATP	recombinant wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Homo sapiens

General Information

General Information	Comment	Organism
additional information	modeling of the catalytic mechanism, overview. Residue Asp81 plays a critical role in the phosphoryl transfer step, but not in the binding of ATP	Homo sapiens
physiological function	lipid kinase sphingosine kinase 1 catalyzes the conversion of sphingosine to sphingosine-1-phosphate, which plays a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the sphingosine-1-phosphate levels in cells, the enzyme is an important regulator for diverse cellular functions	Homo sapiens