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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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Location of the conserved HP and D/E-D/E-G motifs in the P. multocida sialyltransferase PmST1. A. Surface representation of PmST1 (PDB entry 2EX1). The motifs are coloured in red (D/E-D/E-G) and blue (HP) while bound CMP is shown in yellow stick representation. B. Active site view of PmST1. Amino acids that are part of the two motifs are labelled and depicted in green stick representation. Hydrogen bonds are shown as dotted line while the bound donor analogue CMP-3F(α)-Neu5Ac is depicted in yellow and the bound acceptor lactose is shown in cyan. Figures were generated with Pymol (http://www.pymol.org).
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fig11: Location of the conserved HP and D/E-D/E-G motifs in the P. multocida sialyltransferase PmST1. A. Surface representation of PmST1 (PDB entry 2EX1). The motifs are coloured in red (D/E-D/E-G) and blue (HP) while bound CMP is shown in yellow stick representation. B. Active site view of PmST1. Amino acids that are part of the two motifs are labelled and depicted in green stick representation. Hydrogen bonds are shown as dotted line while the bound donor analogue CMP-3F(α)-Neu5Ac is depicted in yellow and the bound acceptor lactose is shown in cyan. Figures were generated with Pymol (http://www.pymol.org).

Mentions: Because NmB-polySTs carrying the point mutations H278A and P279A retained residual activity in vitro, kinetic properties of these mutants could be determined. Both mutants were expressed as MBP fusion proteins, purified and tested in the enzyme-linked polyST assay. As listed in Table 1, Vmax values for CMP-Neu5Ac were decreased by a factor of 4 (P279A) and 6 (H278A) with respect to the wild-type enzyme and, in both proteins, Km values for CMP-Neu5Ac were increased three- to fivefold. These data suggest that the HP motif of NmB-polyST is involved in binding of the donor substrate CMP-Neu5Ac. To further analyse the effect of both mutations on acceptor binding, Km values for colominic acid were determined. Interestingly, Michaelis constants were not significantly influenced by either mutation indicating that: (i) the HP motif does not vitally participate in acceptor binding, and (ii) the introduced point mutations did not cause major structural changes as the acceptor binding site appeared to be largely unaffected. Remarkably, the recently solved crystal structure of P. multocida sialyltransferase PmST1 (Ni et al., 2006) revealed a similar CMP-Neu5Ac binding function of the HP motif in the GT-80 sialyltransferase family. The enzyme consists of two Rossmann domains that form a deep cleft in which the active site is located and has been crystallized in the presence and absence of donor and acceptor substrates. The active site of PmST1 with bound donor analogue CMP-3F(α)Neu5Ac and acceptor lactose (Ni et al., 2007) is shown in Fig. 11 and illustrates that the histidine residue of the HP motif (H311 in PmST1) is directly involved in CMP-Neu5Ac binding. It forms hydrogen bonds with the phosphate group of CMP and with the carboxylate function of the sialic acid moiety. Moreover, this structure shows that also the D/E-D/E-G motif is located directly at the active site cleft of PmST1 (DDG) and is involved in binding of donor and acceptor substrates. The second aspartic acid residue of the DDG sequence forms hydrogen bonds to the acceptor lactose and to the hydroxyl group in position C4 of the sialic acid moiety. In combination, the structural and biochemical data provide strong evidence that the D/E-D/E-G and HP sequences are crucial for CMP-Neu5Ac binding and enzyme catalysis, in bacterial sialyl- and polysialyltransferases that harbour these motifs.


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)

Location of the conserved HP and D/E-D/E-G motifs in the P. multocida sialyltransferase PmST1. A. Surface representation of PmST1 (PDB entry 2EX1). The motifs are coloured in red (D/E-D/E-G) and blue (HP) while bound CMP is shown in yellow stick representation. B. Active site view of PmST1. Amino acids that are part of the two motifs are labelled and depicted in green stick representation. Hydrogen bonds are shown as dotted line while the bound donor analogue CMP-3F(α)-Neu5Ac is depicted in yellow and the bound acceptor lactose is shown in cyan. Figures were generated with Pymol (http://www.pymol.org).
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Related In: Results  -  Collection

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

fig11: Location of the conserved HP and D/E-D/E-G motifs in the P. multocida sialyltransferase PmST1. A. Surface representation of PmST1 (PDB entry 2EX1). The motifs are coloured in red (D/E-D/E-G) and blue (HP) while bound CMP is shown in yellow stick representation. B. Active site view of PmST1. Amino acids that are part of the two motifs are labelled and depicted in green stick representation. Hydrogen bonds are shown as dotted line while the bound donor analogue CMP-3F(α)-Neu5Ac is depicted in yellow and the bound acceptor lactose is shown in cyan. Figures were generated with Pymol (http://www.pymol.org).
Mentions: Because NmB-polySTs carrying the point mutations H278A and P279A retained residual activity in vitro, kinetic properties of these mutants could be determined. Both mutants were expressed as MBP fusion proteins, purified and tested in the enzyme-linked polyST assay. As listed in Table 1, Vmax values for CMP-Neu5Ac were decreased by a factor of 4 (P279A) and 6 (H278A) with respect to the wild-type enzyme and, in both proteins, Km values for CMP-Neu5Ac were increased three- to fivefold. These data suggest that the HP motif of NmB-polyST is involved in binding of the donor substrate CMP-Neu5Ac. To further analyse the effect of both mutations on acceptor binding, Km values for colominic acid were determined. Interestingly, Michaelis constants were not significantly influenced by either mutation indicating that: (i) the HP motif does not vitally participate in acceptor binding, and (ii) the introduced point mutations did not cause major structural changes as the acceptor binding site appeared to be largely unaffected. Remarkably, the recently solved crystal structure of P. multocida sialyltransferase PmST1 (Ni et al., 2006) revealed a similar CMP-Neu5Ac binding function of the HP motif in the GT-80 sialyltransferase family. The enzyme consists of two Rossmann domains that form a deep cleft in which the active site is located and has been crystallized in the presence and absence of donor and acceptor substrates. The active site of PmST1 with bound donor analogue CMP-3F(α)Neu5Ac and acceptor lactose (Ni et al., 2007) is shown in Fig. 11 and illustrates that the histidine residue of the HP motif (H311 in PmST1) is directly involved in CMP-Neu5Ac binding. It forms hydrogen bonds with the phosphate group of CMP and with the carboxylate function of the sialic acid moiety. Moreover, this structure shows that also the D/E-D/E-G motif is located directly at the active site cleft of PmST1 (DDG) and is involved in binding of donor and acceptor substrates. The second aspartic acid residue of the DDG sequence forms hydrogen bonds to the acceptor lactose and to the hydroxyl group in position C4 of the sialic acid moiety. In combination, the structural and biochemical data provide strong evidence that the D/E-D/E-G and HP sequences are crucial for CMP-Neu5Ac binding and enzyme catalysis, in bacterial sialyl- and polysialyltransferases that harbour these motifs.

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