Literature summary for 2.3.1.50 extracted from

Beattie, A.E.; Gupta, S.D.; Frankova, L.; Kazlauskaite, A.; Harmon, J.M.; Dunn, T.M.; Campopiano, D.J.
The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT): effects of the small subunits and insights from bacterial mimics of human hLCB2a HSAN1 mutations (2013), BioMed Res. Int., 2013, 194371.

Activating Compound

Activating Compound	Comment	Organism	Structure
small subunits of serine palmitoyltransferase	small subunits of SPT, ssSPTa and ssSPTb, increase human SPT activity by 50-100fold when coexpressed with enzyme subunits hLCB1 and hLCBa	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Sphingomonas paucimobilis
recombinant expression of subunit LCB2a mutants in HEK293 cells, recombinant His-tagged enzyme expression in Escherichia coli strain BL21(DE3). Recombinant expression of wild-type hLCB1/hLCB2a subunits in Saccharomyces cerevisiae microsomes in the absence of either small subunit, the enzyme shows very low activity. Coexpression of each of the three subunit hLCB2a mutants, V359M, G382V, and I504F, along with subunit hLCB1 results in low activity, with G382V barely detectable above background	Homo sapiens

Cloned (Comment)

Organism

recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)

Sphingomonas paucimobilis

recombinant expression of subunit LCB2a mutants in HEK293 cells, recombinant His-tagged enzyme expression in Escherichia coli strain BL21(DE3). Recombinant expression of wild-type hLCB1/hLCB2a subunits in Saccharomyces cerevisiae microsomes in the absence of either small subunit, the enzyme shows very low activity. Coexpression of each of the three subunit hLCB2a mutants, V359M, G382V, and I504F, along with subunit hLCB1 results in low activity, with G382V barely detectable above background

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
C133W	naturally occuring mutation in subunit LCB1 involved in hereditary sensory and autonomic neuropathy type I disease, the mutant shows reduced activity compared tot he wild-type enzyme	Homo sapiens
C133Y	naturally occuring mutation in subunit LCB1 involved in hereditary sensory and autonomic neuropathy type I disease, the mutant shows reduced activity compared tot he wild-type enzyme	Homo sapiens
G268V	site-directed mutagenesis, the mutation perturbs the pyridoxal 5'-phosphate cofactor binding and reduces the affinity for both substrates, inactive mutant, the protein is expressed in a completely insoluble form, structure homology modeling of the mutant enzyme using the Sp SPT PLP-L-serine external aldimine structure, PDB ID 2W8J	Sphingomonas paucimobilis
G382V	naturally occuring mutation in subunit LCB2a involved in hereditary sensory and autonomic neuropathy type I disease, the activity of mutant G382V is barely detectable above background	Homo sapiens
G385F	site-directed mutagenesis, the mutation perturbs the pyridoxal 5'-phosphate cofactor binding, reduces the affinity for both substrates, decreases the enzyme activity, soluble protein	Sphingomonas paucimobilis
I504F	naturally occuring mutation in subunit LCB2a involved in hereditary sensory and autonomic neuropathy type I disease, the mutant shows reduced activity compared tot he wild-type enzyme	Homo sapiens
additional information	an enzyme knockout strain, which completely lacks SPT activity, is not viable unless supplemented with a long chain base or a competent, active SPT complex. Coexpression of each of the three subunit hLCB2a mutants, V359M, G382V, and I504F, along with subunit hLCB1 results in low activity, with G382V barely detectable above background. When small subunit ssSPTb is expressed, heterodimers containing the G382V and I504F mutant hLCB2a subunits are activated to the same extent as wild-type heterodimers, but heterodimers containing the V359Mmutant subunit are less well activated	Homo sapiens
V246M	site-directed mutagenesis, the mutation perturbs the pyridoxal 5'-phosphate cofactor binding, reduces the affinity for both substrates, decreases the enzyme activity, soluble protein	Sphingomonas paucimobilis
V359M	naturally occuring mutation in subunit LCB2a involved in hereditary sensory and autonomic neuropathy type I disease, the mutant shows reduced activity compared tot he wild-type enzyme	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics of wild-type and mutant enzymes	Sphingomonas paucimobilis
additional information	-	additional information	Michaelis-Menten kinetics of wild-type and mutant enzymes	Homo sapiens
0.0356	-	palmitoyl-CoA	recombinant wild-type enzyme, pH 7.5, 25°C	Sphingomonas paucimobilis
0.0522	-	palmitoyl-CoA	recombinant mutant G385F, pH 7.5, 25°C	Sphingomonas paucimobilis
0.128	-	palmitoyl-CoA	recombinant mutant V246M, pH 7.5, 25°C	Sphingomonas paucimobilis
1.6	-	L-serine	recombinant wild-type enzyme, pH 7.5, 25°C	Sphingomonas paucimobilis
2.5	-	L-serine	recombinant mutant V246M, pH 7.5, 25°C	Sphingomonas paucimobilis
3	-	L-serine	recombinant mutant G385F, pH 7.5, 25°C	Sphingomonas paucimobilis

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
membrane	membrane-bound enzyme	Homo sapiens	16020	-
microsome	-	Homo sapiens	-	-

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
90000	-	about, recombinant wild-type enzyme and mutants V246M and G385F, gel filtration	Sphingomonas paucimobilis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
palmitoyl-CoA + L-serine	Sphingomonas paucimobilis	-	CoA + 3-dehydro-D-sphinganine + CO2	-	?
palmitoyl-CoA + L-serine	Homo sapiens	-	CoA + 3-dehydro-D-sphinganine + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	O15269 and O15270	subunits hLCB1 (O15269) and hLCB2a/b (O15270)	-
Sphingomonas paucimobilis	Q93UV0	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Homo sapiens
recombinant His-tagged wild-type and enzyme and mutants V246M and G385F from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Sphingomonas paucimobilis

Reaction

Reaction	Comment	Organism	Reaction ID
palmitoyl-CoA + L-serine = CoA + 3-dehydro-D-sphinganine + CO2	the reaction proceeds via a key carbanion/quinonoid species and an internal aldimine/PLP-bound form of the enzyme	Sphingomonas paucimobilis	a
palmitoyl-CoA + L-serine = CoA + 3-dehydro-D-sphinganine + CO2	the reaction proceeds via a key carbanion/quinonoid species and an internal aldimine/PLP-bound form of the enzyme	Homo sapiens	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
palmitoyl-CoA + L-serine	-	Sphingomonas paucimobilis	CoA + 3-dehydro-D-sphinganine + CO2	-	?
palmitoyl-CoA + L-serine	-	Homo sapiens	CoA + 3-dehydro-D-sphinganine + CO2	-	?
S-(2-oxoheptadecyl)-CoA + L-serine	-	Homo sapiens	CoA + ?	-	?

Subunits

Subunits	Comment	Organism
heterodimer	the membrane-bound heterodimer composed of two subunits hLCB1 and hLCB2a/b	Sphingomonas paucimobilis
heterodimer	the membrane-bound heterodimer composed of two subunits hLCB1 and hLCB2a/b. The hLCB1 and hLCB2 subunits display relatively high sequence homology to each other, but only the hLCB2a subunit contains key catalytic residues including the lysine that binds pyridoxal 5'-phosphate. Subunit hLCB1 lacks these residues but contains other residues involved in catalysis,the hLCB1/hLCB2a heterodimer has a single active site	Homo sapiens

Synonyms

Synonyms	Comment	Organism
serine palmitoyltransferase	-	Sphingomonas paucimobilis
serine palmitoyltransferase	-	Homo sapiens
SPT	-	Sphingomonas paucimobilis
SPT	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Sphingomonas paucimobilis
25	-	assay at	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.41	-	L-serine	recombinant mutant G385F, pH 7.5, 25°C	Sphingomonas paucimobilis
0.41	-	palmitoyl-CoA	recombinant mutant G385F, pH 7.5, 25°C	Sphingomonas paucimobilis
0.44	-	L-serine	recombinant mutant V246M, pH 7.5, 25°C	Sphingomonas paucimobilis
0.44	-	palmitoyl-CoA	recombinant mutant V246M, pH 7.5, 25°C	Sphingomonas paucimobilis
1.14	-	L-serine	recombinant wild-type enzyme, pH 7.5, 25°C	Sphingomonas paucimobilis
1.14	-	palmitoyl-CoA	recombinant wild-type enzyme, pH 7.5, 25°C	Sphingomonas paucimobilis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Sphingomonas paucimobilis
7.5	-	assay at	Homo sapiens

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	dependent on	Sphingomonas paucimobilis
pyridoxal 5'-phosphate	dependent on, bound by lysine 265 residue on subunit LCB2a	Homo sapiens

General Information

General Information	Comment	Organism
malfunction	mutations in both subunits hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) are identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Structure homology modeling to understand the impact of the hLCB2a mutations on the mechanism of the enzyme using the structure data from the Sphingomonas paucimobilis enzyme	Homo sapiens
metabolism	the enzyme catalyses the first step of de novo sphingolipid biosynthesis	Sphingomonas paucimobilis
metabolism	the enzyme catalyses the first step of de novo sphingolipid biosynthesis	Homo sapiens
additional information	structure modeling	Sphingomonas paucimobilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.136	-	palmitoyl-CoA	recombinant mutant V246M, pH 7.5, 25°C	Sphingomonas paucimobilis
0.176	-	L-serine	recombinant mutant V246M, pH 7.5, 25°C	Sphingomonas paucimobilis
0.712	-	L-serine	recombinant wild-type enzyme, pH 7.5, 25°C	Sphingomonas paucimobilis
3.437	-	L-serine	recombinant mutant G385F, pH 7.5, 25°C	Sphingomonas paucimobilis
7.854	-	palmitoyl-CoA	recombinant mutant G385F, pH 7.5, 25°C	Sphingomonas paucimobilis
32.02	-	palmitoyl-CoA	recombinant wild-type enzyme, pH 7.5, 25°C	Sphingomonas paucimobilis