Cloned (Comment) | Organism |
---|---|
expression of trigger factor-fused SPS2 in Escherichia coli | Aquifex aeolicus |
Crystallization (Comment) | Organism |
---|---|
SPS2 free or with bound alpha/beta-methylene ATP, X-ray diffraction structure determination and analysis at 1.98-2.1 A resolution, molecular replacement | Aquifex aeolicus |
Protein Variants | Comment | Organism |
---|---|---|
C13S | site-directed mutagenesis, inactive mutant, the activity cannot be recovered by the NifS-like protein | Aquifex aeolicus |
C17S | site-directed mutagenesis, inactive mutant, the activity can be recovered by the NifS-like protein | Aquifex aeolicus |
K16A | site-directed mutagenesis, inactive mutant | Aquifex aeolicus |
N79A | site-directed mutagenesis, the mutation enhances the ATP consumption by about 3fold compared to the wild-type enzyme | Aquifex aeolicus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | ATP binding, metal binding, and the formation of their binding sites are interdependent, Thr221 and Gly222 are involved in metal binding | Aquifex aeolicus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + selenide + H2O | Aquifex aeolicus | Sec replaces the catalytically essential Cys residue, SPS is strictly conserved in the organisms that utilize Sec and/or selenouridine | AMP + selenophosphate + phosphate | - |
? | |
additional information | Aquifex aeolicus | SPS orthologs, SPS1 and SPS2, exist, and SPS2 is the one that synthesizes selenophosphate, while SPS1 does not | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aquifex aeolicus | O67139 | gene selD | - |
Purification (Comment) | Organism |
---|---|
recombinant trigger factor-fused SPS2 from Escherichia coli, during purification, the N-terminal trigger factor portion is removed by proteolysis with thrombin, and the final SPS protein comprises the full-length SPS region, residues 1-336, and nine extra N-terminal residues, GSGGIEGRH, which are derived from the linker | Aquifex aeolicus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + selenide + H2O = AMP + selenophosphate + phosphate | SPS catalyzes the exchange of the ATP gamma-phosphate group in the absence of selenide, suggesting that the beta-gamma-phosphate bond, but not the alpha-beta-phosphate bond, could be the initial cleavage site, formation of a phosphorylated enzyme intermediate, catalytic mechanism, overview | Aquifex aeolicus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + selenide + H2O | Sec replaces the catalytically essential Cys residue, SPS is strictly conserved in the organisms that utilize Sec and/or selenouridine | Aquifex aeolicus | AMP + selenophosphate + phosphate | - |
? | |
ATP + selenide + H2O | the phosphate moiety of selenophosphate is derived from the ATP gamma-phosphate group, while the orthophosphate is from the beta-phosphate group. Although Thr221 is not essential for the reaction, it contributes to the enzyme activity, probably by interacting with the ATP gamma-phosphate group and a water molecule. Sec/Cys13 and Lys16 are essential for the SPS catalysis. Thr221 and Gly222 interact with the gamma-phosphate group of ATP | Aquifex aeolicus | AMP + selenophosphate + phosphate | - |
? | |
additional information | SPS orthologs, SPS1 and SPS2, exist, and SPS2 is the one that synthesizes selenophosphate, while SPS1 does not | Aquifex aeolicus | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
More | SPS belongs to the PurM superfamily | Aquifex aeolicus |
selenophosphate synthase | - |
Aquifex aeolicus |
SPS | - |
Aquifex aeolicus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
alpha,beta-methylene ATP | i.e. MPCPP, binding structure, overview | Aquifex aeolicus | |
ATP | the ATP-binding site is formed at the subunit interface of the homodimer. Four Asp residues coordinate four metal ions to bind the phosphate groups of AMPCPP. In the free SPS structure, the two loop regions in the ATP-binding site are not ordered, and no enzyme-associated metal is observed. This suggests that ATP binding, metal binding, and the formation of their binding sites are interdependent, Thr221 and Gly222 interact with the gamma-phosphate group of ATP | Aquifex aeolicus |