Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Sphingomonas paucimobilis |
Crystallization (Comment) | Organism |
---|---|
determination of the crystal structure of enzyme mutant K265A that diffract to 1.6 A resolution and contain a canonical dimer in the asymmetric unit. Crystallization of the wild-type SPT:PLP-myriocin aldimine complex is not possible, most likely due to aldimine degradation | Sphingomonas paucimobilis |
Protein Variants | Comment | Organism |
---|---|---|
K265A | site-directed mutagenesis, the mutant is unable to bind pyridoxal 5'-phosphate, structure of a SPT K265A:PLP-myriocin external aldimine complex, molecular replacement study | Sphingomonas paucimobilis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
myriocin | i.e. (2S,3R,4R,6E)-2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxo-6-eicosenoic acid, or thermozymocidin and ISP-1, is a fungal natural product, kinetics and molecular mechanism of enzyme inhibition, overview. Myriocin is a potent SPT enzyme inhibitor. It initially forms an external aldimine with pyridoxal 5'-phosphate at the active site, the structure of the resulting co-complex explains its nanomolar affinity for the enzyme. The cofactor-inhibitor co-complex, PLP-myriocin aldimine, catalytically degrades via an unexpected retro-aldol-like cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of the enzyme by covalent modification of the essential catalytic lysine. This dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition. Incubations of enzyme SPT with myriocin is consistent with the formation of an initial enzyme-inhibitor complex which is noncovalent in nature and reversible, albeit with a very slow off rate (koff), the PLP-myriocin aldimine as the initial competitive inhibitory species. In contrast, for the enzyme preincubated with myriocin for 16 h, no detectable regain in activity is observed after dialysis either at 3 or 24 h, the second formed covalent adduct acts as an irreversible inhibitor of enzyme SPT | Sphingomonas paucimobilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics of wild-type and mutant enzyme | Sphingomonas paucimobilis |
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 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Sphingomonas paucimobilis | Q93UV0 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Sphingomonas paucimobilis |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
palmitoyl-CoA + L-serine = CoA + 3-dehydro-D-sphinganine + CO2 | pyridoxal 5'-phosphate dependent reaction mechanism, key active site residues are His159, Asp231, His234, and Lys265 | Sphingomonas paucimobilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
palmitoyl-CoA + L-serine | - |
Sphingomonas paucimobilis | CoA + 3-dehydro-D-sphinganine + CO2 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
serine palmitoyltransferase | - |
Sphingomonas paucimobilis |
SPT | - |
Sphingomonas paucimobilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Sphingomonas paucimobilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | 8 | assay at | Sphingomonas paucimobilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | dependent on | Sphingomonas paucimobilis |
General Information | Comment | Organism |
---|---|---|
metabolism | sphingolipids are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal 5'-phosphate-dependent enzyme serine palmitoyltransferase | Sphingomonas paucimobilis |
additional information | key active site residues are His159, Asp231, His234, and Lys265 | Sphingomonas paucimobilis |