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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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Conserved motifs in bacterial sialyl- and polysialyltransferases. Sequence alignment of bacterial sialyltransferases identifying two short motifs conserved in CAZy families GT-38 (bacterial polysialyltransferases), GT-52 (bacterial LOS-sialyltransferases) and in pfam family 05855 (similar to H. ducreyi sialyltransferase Lst). To improve clarity of the illustration only three representatives of pfam 05855 are shown. PolyST_NmB (AAA20478), polyST_EcK92 (AAA24215), polyST_EcK1 (CAA43053), 2,3ST_cpsK_Sa (EAO062164), 2,3-ST_Hd (AAD28703), SiaA_Lst_Hi (AAL38659), PM0508_Lst_Pm (AAK02592), LsgB_Hi (AAX88755), Cps8K_Sa (AAR29926), 2,3_ST_Nm (AAC44544), lst_Ng (AAY41933), lst_Ap (AAS66624), Psyc_0663_Pa (AAZ18522). Alignments were made using Multalign (Corpet, 1988).
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fig08: Conserved motifs in bacterial sialyl- and polysialyltransferases. Sequence alignment of bacterial sialyltransferases identifying two short motifs conserved in CAZy families GT-38 (bacterial polysialyltransferases), GT-52 (bacterial LOS-sialyltransferases) and in pfam family 05855 (similar to H. ducreyi sialyltransferase Lst). To improve clarity of the illustration only three representatives of pfam 05855 are shown. PolyST_NmB (AAA20478), polyST_EcK92 (AAA24215), polyST_EcK1 (CAA43053), 2,3ST_cpsK_Sa (EAO062164), 2,3-ST_Hd (AAD28703), SiaA_Lst_Hi (AAL38659), PM0508_Lst_Pm (AAK02592), LsgB_Hi (AAX88755), Cps8K_Sa (AAR29926), 2,3_ST_Nm (AAC44544), lst_Ng (AAY41933), lst_Ap (AAS66624), Psyc_0663_Pa (AAZ18522). Alignments were made using Multalign (Corpet, 1988).

Mentions: To identify amino acid residues critical for (poly)sialyltransferase activity, we searched for common sequence motifs in the available bacterial sialyltransferase sequences. By iterative steps of pairwise and multiple alignments combined with visual inspection of the sequences, we identified two short motifs (D/E-D/E-G and HP). Both motifs are present in a range of enzymes with otherwise little identity (Fig. 8) that, based on functional and sequence properties, had been allocated to different CAZy families (GT-38 and GT-52) and to pfam family 05855. While GT-38 contains bacterial polysialyltransferases, GT-52 includes bacterial LOS sialyltransferases and pfam 05855 groups sialyltransferases similar to the sialyltransferase Lst of Haemophilus ducreyi (Bozue et al., 1999). The D/E-D/E-G and the HP motif identified in this study are conserved in all members of the three families and, interestingly, are also found in the bacterial LOS sialyltransferases grouped in GT-80. However, the relation to GT-80 family members is not easily seen in an alignment, as the stretch of sequence between the D/E-D/E-G and HP motif is approximately 50 amino acids longer than in the other families.


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)

Conserved motifs in bacterial sialyl- and polysialyltransferases. Sequence alignment of bacterial sialyltransferases identifying two short motifs conserved in CAZy families GT-38 (bacterial polysialyltransferases), GT-52 (bacterial LOS-sialyltransferases) and in pfam family 05855 (similar to H. ducreyi sialyltransferase Lst). To improve clarity of the illustration only three representatives of pfam 05855 are shown. PolyST_NmB (AAA20478), polyST_EcK92 (AAA24215), polyST_EcK1 (CAA43053), 2,3ST_cpsK_Sa (EAO062164), 2,3-ST_Hd (AAD28703), SiaA_Lst_Hi (AAL38659), PM0508_Lst_Pm (AAK02592), LsgB_Hi (AAX88755), Cps8K_Sa (AAR29926), 2,3_ST_Nm (AAC44544), lst_Ng (AAY41933), lst_Ap (AAS66624), Psyc_0663_Pa (AAZ18522). Alignments were made using Multalign (Corpet, 1988).
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Related In: Results  -  Collection

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fig08: Conserved motifs in bacterial sialyl- and polysialyltransferases. Sequence alignment of bacterial sialyltransferases identifying two short motifs conserved in CAZy families GT-38 (bacterial polysialyltransferases), GT-52 (bacterial LOS-sialyltransferases) and in pfam family 05855 (similar to H. ducreyi sialyltransferase Lst). To improve clarity of the illustration only three representatives of pfam 05855 are shown. PolyST_NmB (AAA20478), polyST_EcK92 (AAA24215), polyST_EcK1 (CAA43053), 2,3ST_cpsK_Sa (EAO062164), 2,3-ST_Hd (AAD28703), SiaA_Lst_Hi (AAL38659), PM0508_Lst_Pm (AAK02592), LsgB_Hi (AAX88755), Cps8K_Sa (AAR29926), 2,3_ST_Nm (AAC44544), lst_Ng (AAY41933), lst_Ap (AAS66624), Psyc_0663_Pa (AAZ18522). Alignments were made using Multalign (Corpet, 1988).
Mentions: To identify amino acid residues critical for (poly)sialyltransferase activity, we searched for common sequence motifs in the available bacterial sialyltransferase sequences. By iterative steps of pairwise and multiple alignments combined with visual inspection of the sequences, we identified two short motifs (D/E-D/E-G and HP). Both motifs are present in a range of enzymes with otherwise little identity (Fig. 8) that, based on functional and sequence properties, had been allocated to different CAZy families (GT-38 and GT-52) and to pfam family 05855. While GT-38 contains bacterial polysialyltransferases, GT-52 includes bacterial LOS sialyltransferases and pfam 05855 groups sialyltransferases similar to the sialyltransferase Lst of Haemophilus ducreyi (Bozue et al., 1999). The D/E-D/E-G and the HP motif identified in this study are conserved in all members of the three families and, interestingly, are also found in the bacterial LOS sialyltransferases grouped in GT-80. However, the relation to GT-80 family members is not easily seen in an alignment, as the stretch of sequence between the D/E-D/E-G and HP motif is approximately 50 amino acids longer than in the other families.

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