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Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases.

Freiberger F, Claus H, Günzel A, Oltmann-Norden I, Vionnet J, Mühlenhoff M, Vogel U, Vann WF, Gerardy-Schahn R, Stummeyer K - Mol. Microbiol. (2007)

Bottom Line: The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST).Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1.Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

ABSTRACT
The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. 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. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

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Enzymatic characterization of NmB-polyST. A. To analyse acceptor specificity, oligomeric α2,8-linked sialic acids with a degree of polymerization (DP) ranging from DP2 to DP6 (0.5 mM) were assayed at constant enzyme (4 μM) and CMP-Neu5Ac concentrations (1 mM) using the continuous spectrophotometric assay. PolyST activity is detected as decreasing absorption at 340 nm (NADH oxidation). B. To determine Km and Vmax of NmB-polyST for the donor substrate CMP-Neu5Ac, measurements were performed at 30°C and 0.28 mg ml−1 colominic acid. The resulting substrate velocity curve and Lineweaver–Burk plot are depicted. Kinetic parameters were obtained by non-linear regression in Prism (GraphPad Software).
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fig10: Enzymatic characterization of NmB-polyST. A. To analyse acceptor specificity, oligomeric α2,8-linked sialic acids with a degree of polymerization (DP) ranging from DP2 to DP6 (0.5 mM) were assayed at constant enzyme (4 μM) and CMP-Neu5Ac concentrations (1 mM) using the continuous spectrophotometric assay. PolyST activity is detected as decreasing absorption at 340 nm (NADH oxidation). B. To determine Km and Vmax of NmB-polyST for the donor substrate CMP-Neu5Ac, measurements were performed at 30°C and 0.28 mg ml−1 colominic acid. The resulting substrate velocity curve and Lineweaver–Burk plot are depicted. Kinetic parameters were obtained by non-linear regression in Prism (GraphPad Software).

Mentions: To compare wild-type and mutant polySTs with partial activity (particularly mutants H278A and P279A) we decided to perform kinetic studies. As a first step towards this goal, the glycosyltransferase assay developed by Gosselin et al. (1994) was adapted to assay polyST activity. This spectrophotometric assay links the release of CMP by polyST during polySia synthesis to NADH oxidation and thus enables continuous monitoring of the enzyme reaction. PolyST activity towards short oligosialic acid acceptors (DP2 to DP5) was measured at constant CMP-Neu5Ac and enzyme concentrations. As shown in Fig. 10A, efficient chain elongation required oligomers of at least DP3. These findings are in agreement with the acceptor dependence obtained when polyST-containing membrane preparations of E. coli K1 or K92 were assayed in radiochemical or HPLC-based test systems (Steenbergen and Vimr, 1990; Ferrero et al., 1991; Chao et al., 1999).


Biochemical characterization of a Neisseria meningitidis polysialyltransferase reveals novel functional motifs in bacterial sialyltransferases.

Freiberger F, Claus H, Günzel A, Oltmann-Norden I, Vionnet J, Mühlenhoff M, Vogel U, Vann WF, Gerardy-Schahn R, Stummeyer K - Mol. Microbiol. (2007)

Enzymatic characterization of NmB-polyST. A. To analyse acceptor specificity, oligomeric α2,8-linked sialic acids with a degree of polymerization (DP) ranging from DP2 to DP6 (0.5 mM) were assayed at constant enzyme (4 μM) and CMP-Neu5Ac concentrations (1 mM) using the continuous spectrophotometric assay. PolyST activity is detected as decreasing absorption at 340 nm (NADH oxidation). B. To determine Km and Vmax of NmB-polyST for the donor substrate CMP-Neu5Ac, measurements were performed at 30°C and 0.28 mg ml−1 colominic acid. The resulting substrate velocity curve and Lineweaver–Burk plot are depicted. Kinetic parameters were obtained by non-linear regression in Prism (GraphPad Software).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169525&req=5

fig10: Enzymatic characterization of NmB-polyST. A. To analyse acceptor specificity, oligomeric α2,8-linked sialic acids with a degree of polymerization (DP) ranging from DP2 to DP6 (0.5 mM) were assayed at constant enzyme (4 μM) and CMP-Neu5Ac concentrations (1 mM) using the continuous spectrophotometric assay. PolyST activity is detected as decreasing absorption at 340 nm (NADH oxidation). B. To determine Km and Vmax of NmB-polyST for the donor substrate CMP-Neu5Ac, measurements were performed at 30°C and 0.28 mg ml−1 colominic acid. The resulting substrate velocity curve and Lineweaver–Burk plot are depicted. Kinetic parameters were obtained by non-linear regression in Prism (GraphPad Software).
Mentions: To compare wild-type and mutant polySTs with partial activity (particularly mutants H278A and P279A) we decided to perform kinetic studies. As a first step towards this goal, the glycosyltransferase assay developed by Gosselin et al. (1994) was adapted to assay polyST activity. This spectrophotometric assay links the release of CMP by polyST during polySia synthesis to NADH oxidation and thus enables continuous monitoring of the enzyme reaction. PolyST activity towards short oligosialic acid acceptors (DP2 to DP5) was measured at constant CMP-Neu5Ac and enzyme concentrations. As shown in Fig. 10A, efficient chain elongation required oligomers of at least DP3. These findings are in agreement with the acceptor dependence obtained when polyST-containing membrane preparations of E. coli K1 or K92 were assayed in radiochemical or HPLC-based test systems (Steenbergen and Vimr, 1990; Ferrero et al., 1991; Chao et al., 1999).

Bottom Line: The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST).Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1.Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

View Article: PubMed Central - PubMed

Affiliation: Abteilung Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

ABSTRACT
The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. 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. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.

Show MeSH
Related in: MedlinePlus