Literature summary for 2.5.1.22 extracted from

MacLean, H.E.; Chiu, W.S.; Notini, A.J.; Axell, A.; Davey, R.A.; McManus, J.F.; Ma, C.; Plant, D.R.; Lynch, G.S.; Zajac, J.D.
Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice (2008), FASEB J., 22, 2676-2689.

Cloned(Commentary)

Cloned (Comment)	Organism
DNA fragment encoding human enzyme is amplified by PCR and subcloned into the pET28a-LIC vector downstream of the polyhistidine coding region. Enzyme is expressed in the Escherichia coli BL21-Codon Plus(DE3)-RIL strain by the addition of 1 mM isopropyl1-thio-beta-D-galactopyranoside. The N-terminally truncated SpmSyn mutants are generated by PCR and subcloned into the pET28a-LIC vector.	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
Purified SpmSyn is crystallized as ternary complex in the presence of spermidine and 5'-methylthioadenosine or spermidine and 5'-methylthioadenosine using the hanging drop vapor diffusion method. The SpmSyn-5'-methylthioadenosine-spermidine complex is crystallized in 12% polyethylene glycol and 0.1 M MES (pH 6.5). The SpmSyn-5'-methylthioadenosine-spermine complex is crystallized in 18% polyethylene glycol, 0.1 M NaCl, and 0.1 M BisTris (pH 6.5). Crystals are soaked in the corresponding mother liquor supplemented with 20% glycerol as cryoprotectant before freezing in liquid nitrogen.	Homo sapiens

Crystallization (Comment)

Organism

Purified SpmSyn is crystallized as ternary complex in the presence of spermidine and 5'-methylthioadenosine or spermidine and 5'-methylthioadenosine using the hanging drop vapor diffusion method. The SpmSyn-5'-methylthioadenosine-spermidine complex is crystallized in 12% polyethylene glycol and 0.1 M MES (pH 6.5). The SpmSyn-5'-methylthioadenosine-spermine complex is crystallized in 18% polyethylene glycol, 0.1 M NaCl, and 0.1 M BisTris (pH 6.5). Crystals are soaked in the corresponding mother liquor supplemented with 20% glycerol as cryoprotectant before freezing in liquid nitrogen.

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D201A	mutation of Asp201 to Ala decreases the kcat/Km for decarboxylated S-adenosylmethionine by more than 100000fold	Homo sapiens
D201N	mutation of Asp201 to Asn decreases the kcat/Km for decarboxylated S-adenosylmethionine by more than 100000fold	Homo sapiens
D276N	alteration of this residue reduces the kcat/Km for spermidine by more than 200000fold	Homo sapiens
DELTA1-129	0.02% activity compared to the wild-type enzyme	Homo sapiens
DELTA1-145	no activity	Homo sapiens
DELTA1-19	0.003% activity compared to the wild-type enzyme	Homo sapiens
DELTA1-43	0.0002% activity compared to the wild-type enzyme	Homo sapiens
DELTA1-82	0.00023% activity compared to the wild-type enzyme	Homo sapiens
DELTA347-366	truncation of the protein at position 346 removing the last 20 residues lead to a complete loss of activity	Homo sapiens
DELTA358-366A	smaller truncation of only 9 residues has a smaller effect but still reduced activity by 75%	Homo sapiens
E353Q	mutation of Glu353 to Gln reduces the kcat/Km by 800fold	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
5'-methylthioadenosine	SpmSyn is strongly inhibited by 5'-methylthioadenosine. This inhibition does not have great importance in limiting spermine synthesis in vivo because 5'-methylthioadenosine is normally rapidly degraded by 5'-methylthioadenosine phosphorylase. Inhibition of this enzyme allows 5'-methylthioadenosine to accumulate with deleterious effects on polyamine content.	Homo sapiens
additional information	the spermine synthase-5'-methylthioadenosine structure provides a plausible explanation for the potent inhibition of the reaction by this product and the stronger inhibition of spermine synthase compared with spermidine synthase.	Homo sapiens

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
41000	-	predicted	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Homo sapiens	The predominant polyamines in mammalian cells are spermidine and spermine, these polyamines are made by the sequential addition of aminopropyl groups from decarboxylated S-adenosylmethionine.	?	-	?
S-adenosylmethioninamine + spermidine	Homo sapiens	Polyamine sythesis, addition of a second aminopropyl group to the N-10 position of spermidine. The active site with a bound spermidine molecule contains an Asp276 residue, which is in an ideal position to facilitate the deprotonation of the N-10 amino group of spermidine that attacks the C-atom of the aminopropyl group of decarboxylated S-adenosylmethionine	5'-methylthioadenosine + spermine + H+	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P52788	-	-

Purification (Commentary)

Purification (Comment)	Organism
Cell lysate is loaded onto a HiTrap chelating column charged with Ni2+. The enzyme is eluted with an imidazole gradient (50-250 mM) at pH 8.0. The protein is loaded onto a Superdex 200 column equilibrated with 20 mM Tris-HCl and 150 mM NaCl. Thrombin is added to combined fractions containing SpmSyn to remove the His-tag. The protein is further purified to homogeneity by ion-exchange chromatography.	Homo sapiens

Purification (Comment)

Organism

Cell lysate is loaded onto a HiTrap chelating column charged with Ni2+. The enzyme is eluted with an imidazole gradient (50-250 mM) at pH 8.0. The protein is loaded onto a Superdex 200 column equilibrated with 20 mM Tris-HCl and 150 mM NaCl. Thrombin is added to combined fractions containing SpmSyn to remove the His-tag. The protein is further purified to homogeneity by ion-exchange chromatography.

Homo sapiens

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	deletion of the N-terminal domain leads to a complete loss of spermine synthase activity, suggesting that dimerization may be required for activity	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	The predominant polyamines in mammalian cells are spermidine and spermine, these polyamines are made by the sequential addition of aminopropyl groups from decarboxylated S-adenosylmethionine.	Homo sapiens	?	-	?
S-adenosylmethioninamine + spermidine	Polyamine sythesis, addition of a second aminopropyl group to the N-10 position of spermidine. The active site with a bound spermidine molecule contains an Asp276 residue, which is in an ideal position to facilitate the deprotonation of the N-10 amino group of spermidine that attacks the C-atom of the aminopropyl group of decarboxylated S-adenosylmethionine	Homo sapiens	5'-methylthioadenosine + spermine + H+	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 identical subunits, each monomer has 3 domains: a C-terminal domain, which contains the active site, a central domain made up of 4 beta-strands and an N-terminal domain with structural similarity to S-adenosylmethionine decarboxylase. Dimerization occurs mainly through interactions between the N-terminal domains. The structures provide an outline of the active site and a plausible model for catalysis. The active site is similar to those of spermidine synthases but has a larger substrate-binding pocket able to accommodate longer substrates. Asp201 and 276, conserved in aminopropyltransferases, play a key part in the catalytic mechanism. By separate expression of both domains, enzymes are inactive and possess rigid tertiary structure, suggesting that human SpmSyn is a fusion protein.	Homo sapiens
monomer	deletions of all or part of the N-terminal domain lead to the protein existing as a monomer as determined by gel filtration analysis and to virtually complete loss of activity	Homo sapiens

Synonyms

Synonyms	Comment	Organism
spermine synthase	-	Homo sapiens
SpmSyn	-	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0015	-	spermidine	mutant D201A	Homo sapiens
0.0015	-	spermidine	mutant D201N	Homo sapiens
0.0022	-	spermidine	mutant D276N	Homo sapiens
0.64	-	spermidine	mutant E353Q	Homo sapiens
32	-	spermidine	wild-type enzyme	Homo sapiens

pH Optimum

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

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.0003	-	5'-methylthioadenosine	-	Homo sapiens