EC Number | Application | Comment | Organism |
---|---|---|---|
2.4.3.8 | drug development | basis for design of sialyltransferase-specific drugs | Neisseria meningitidis |
EC Number | Cloned (Comment) | Organism |
---|---|---|
2.4.3.8 | expressed in Escherichia coli BL21(DE3) | Neisseria meningitidis |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.4.3.8 | E153A | inactive, single-point mutation of NmB-polyST by QuickChange site-directed mutagenesis | Neisseria meningitidis |
2.4.3.8 | G154A | inactive, single-point mutation of NmB-polyST by QuickChange site-directed mutagenesis | Neisseria meningitidis |
2.4.3.8 | H278A | nearly inactive, single-point mutation of NmB-polyST by QuickChange site-directed mutagenesis. Vmax value for CMP-Neu5Ac is decreased by a factor 6 with respect to the wild-type enzyme and the Km value for CMP-Neu5Ac is 5fold | Neisseria meningitidis |
2.4.3.8 | H278A/P279A | the H278A and P279A mutants maintain residual activity (below 10% of wild type), when both residues are changed to alanine simultaneously (H278A/P279A) enzyme activity is abolished | Neisseria meningitidis |
2.4.3.8 | additional information | mutational analysis of NmBpolyST, emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1, functional importance of the two functional motifs for enzyme catalysis and CMP-Neu5Ac binding shown | Neisseria meningitidis |
2.4.3.8 | additional information | removal of 23 (DELTA23NmBpolyST) and 33 (DELTA33NmB-polyST) amino acids from the N-terminus has only slight effects on solubility and activity of NmB-polyST. Deletion of the first 64 amino acids (DELTA64NmB-polyST) shifts the majority of the expressed protein to the insoluble fraction and no enzymatic activity is detected in soluble or insoluble fractions. Truncated NmBpolySTs lacking the C-terminal domain either partially (NmB-polySTDELTA22, NmB-polySTDELTA60) or completely (NmB-polySTDELTA94, NmB-polySTDELTA95, NmB-polySTDELTA97): each C-terminal truncation completely abolished enzymatic activity | Neisseria meningitidis |
2.4.3.8 | P279A | nearly inactive, single-point mutation of NmB-polyST by QuickChange site-directed mutagenesis. Vmax value for CMP-Neu5Ac is decreased by a factor of 4 with respect to the wild-type enzyme and the Km values for CMP-Neu5Ac is increased 3fold | Neisseria meningitidis |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.4.3.8 | 0.42 | - |
CMP-Neu5Ac | calculated and determined at constant donor concentration of 1 mM CMPNeu5Ac | Neisseria meningitidis |
EC Number | Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|---|
2.4.3.8 | membrane | membrane association is not essential for enzyme functionality. Analyses performed with crude membrane fractions as enzyme source | Neisseria meningitidis | 16020 | - |
2.4.3.8 | additional information | 50% of the wild-type polyST is soluble and enzymatically active, whereby the detected activity is 3fold higher in the soluble than in the insoluble fraction | Neisseria meningitidis | - |
- |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
2.4.3.8 | 100000 | - |
recombinant MBP-NmB-polyST, affinity chromatography and gel filtration, SDS-PAGE, Western blot analysis | Neisseria meningitidis |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.3.8 | additional information | Neisseria meningitidis | enzyme is able to produce large polymers when it is part of the native capsule biosynthesis complex associated with the inner bacterial membrane | ? | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.4.3.8 | Neisseria meningitidis | - |
serogroup B | - |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.4.3.8 | affinity and size exclusion chromatography | Neisseria meningitidis |
EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|---|
2.4.3.8 | additional information | - |
the specific activity of both fusion proteins is increased 2fold compared with enzymes with short tags | Neisseria meningitidis |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.3.8 | CMP-Neu5Ac + GT3-FCHASE | the trisialylganglioside analogue GT3-FCHASE as artificial acceptor substrate | Neisseria meningitidis | CMP + long polySia chains + ? | - |
? | |
2.4.3.8 | additional information | enzyme is able to produce large polymers when it is part of the native capsule biosynthesis complex associated with the inner bacterial membrane | Neisseria meningitidis | ? | - |
? | |
2.4.3.8 | additional information | synthesizes long polysialic acid chains in a non-processive manner in vitro. PolyST activity towards short oligosialic acid acceptors (DP2 to DP5) is measured at constant CMP-Neu5Ac and enzyme concentrations | Neisseria meningitidis | ? | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
2.4.3.8 | More | subsequent structure-function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Removal of the C-terminal extension present in NmB but not in the homologous Escherichia coli enzymes, completely abolished enzymatic activity, proving it as an essential functional domain. Using site-directed mutagenesis and refined protein alignment strategies, identify two functionally important motifs, which are highly conserved in a number of bacterial (poly)sialyltransferases of otherwise unrelated sequences. T7-polyST, NusA-polyST, MBP-polyST, and Strep II-polyST: NmB-polyST fusion proteins with large fusion partners (MBP, NusA) are used | Neisseria meningitidis |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.4.3.8 | membrane-associated polysialyltransferase | - |
Neisseria meningitidis |
2.4.3.8 | NmB-polyST | - |
Neisseria meningitidis |
2.4.3.8 | polysialyltransferase | - |
Neisseria meningitidis |
2.4.3.8 | polyST | - |
Neisseria meningitidis |
2.4.3.8 | sialyltransferase | - |
Neisseria meningitidis |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
2.4.3.8 | 37 | - |
assay at | Neisseria meningitidis |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.4.3.8 | 8 | - |
assay at | Neisseria meningitidis |