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Literature summary extracted from

  • Freiberger, F.; Claus, H.; Günzel, A.; Oltmann-Norden, I.; Vionnet, J.; Mühlenhoff, M.; Vogel, U.; Vann, W.F.; Gerardy-Schahn, R.; Stummeyer, K.
    Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases (2007), Mol. Microbiol., 65, 1258-1275.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.4.3.8 drug development basis for design of sialyltransferase-specific drugs Neisseria meningitidis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.3.8 expressed in Escherichia coli BL21(DE3) Neisseria meningitidis

Protein Variants

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

KM Value [mM]

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

Localization

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
-
-

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

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 ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.3.8 Neisseria meningitidis
-
 serogroup B
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.3.8 affinity and size exclusion chromatography Neisseria meningitidis

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

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 ?
-
 ?

Subunits

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

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.3.8 37
-
 assay at Neisseria meningitidis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.3.8 8
-
 assay at Neisseria meningitidis