EC Number | Cloned (Comment) | Organism |
---|---|---|
2.5.1.22 | 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 |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
2.5.1.22 | 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 |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.5.1.22 | D201A | mutation of Asp201 to Ala decreases the kcat/Km for decarboxylated S-adenosylmethionine by more than 100000fold | Homo sapiens |
2.5.1.22 | D201N | mutation of Asp201 to Asn decreases the kcat/Km for decarboxylated S-adenosylmethionine by more than 100000fold | Homo sapiens |
2.5.1.22 | D276N | alteration of this residue reduces the kcat/Km for spermidine by more than 200000fold | Homo sapiens |
2.5.1.22 | DELTA1-129 | 0.02% activity compared to the wild-type enzyme | Homo sapiens |
2.5.1.22 | DELTA1-145 | no activity | Homo sapiens |
2.5.1.22 | DELTA1-19 | 0.003% activity compared to the wild-type enzyme | Homo sapiens |
2.5.1.22 | DELTA1-43 | 0.0002% activity compared to the wild-type enzyme | Homo sapiens |
2.5.1.22 | DELTA1-82 | 0.00023% activity compared to the wild-type enzyme | Homo sapiens |
2.5.1.22 | DELTA347-366 | truncation of the protein at position 346 removing the last 20 residues lead to a complete loss of activity | Homo sapiens |
2.5.1.22 | DELTA358-366A | smaller truncation of only 9 residues has a smaller effect but still reduced activity by 75% | Homo sapiens |
2.5.1.22 | E353Q | mutation of Glu353 to Gln reduces the kcat/Km by 800fold | Homo sapiens |
EC Number | Inhibitors | Comment | Organism | Structure |
---|---|---|---|---|
2.5.1.22 | 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 | |
2.5.1.22 | 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 |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
2.5.1.22 | 41000 | - |
predicted | Homo sapiens |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.5.1.22 | 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. | ? | - |
? | |
2.5.1.22 | 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+ | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.5.1.22 | Homo sapiens | P52788 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.5.1.22 | 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 |
EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|---|
2.5.1.22 | 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 |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.5.1.22 | 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 | ? | - |
? | |
2.5.1.22 | 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+ | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
2.5.1.22 | 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 |
2.5.1.22 | 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 |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.5.1.22 | spermine synthase | - |
Homo sapiens |
2.5.1.22 | SpmSyn | - |
Homo sapiens |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.5.1.22 | 0.0015 | - |
spermidine | mutant D201A | Homo sapiens | |
2.5.1.22 | 0.0015 | - |
spermidine | mutant D201N | Homo sapiens | |
2.5.1.22 | 0.0022 | - |
spermidine | mutant D276N | Homo sapiens | |
2.5.1.22 | 0.64 | - |
spermidine | mutant E353Q | Homo sapiens | |
2.5.1.22 | 32 | - |
spermidine | wild-type enzyme | Homo sapiens |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.5.1.22 | 7.5 | - |
assay at | Homo sapiens |
EC Number | Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.5.1.22 | 0.0003 | - |
5'-methylthioadenosine | - |
Homo sapiens |