%0 Journal Article %1 romanow_biochemical_2013 %A Romanow, Angela %A Haselhorst, Thomas %A Stummeyer, Katharina %A Claus, Heike %A Bethe, Andrea %A Mühlenhoff, Martina %A Vogel, Ulrich %A von Itzstein, Mark %A Gerardy-Schahn, Rita %D 2013 %J The Journal of biological chemistry %K Bacterial Bacterial_Capsules Bacterial_Proteins Evolution Gene_Fusion Humans Meningitis Meningococcal Molecular Mutagenesis Neisseria_meningitidis Polysaccharides Protein_Structure Sepsis Serogroup_W-135 Sialyltransferases Site-Directed Tertiary Uridine_Diphosphate_Galactose bacterial_capsules %N 17 %P 11718--11730 %R 10.1074/jbc.M113.452276 %T Biochemical and biophysical characterization of the sialyl-/hexosyltransferase synthesizing the meningococcal serogroup W135 heteropolysaccharide capsule %V 288 %X Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. Crucial virulence determinants of pathogenic Nm strains are the polysaccharide capsules that support invasion by hindering complement attack. In NmW-135 and NmY the capsules are built from the repeating units (→ 6)-α-D-Gal-(1 → 4)-α-Neu5Ac-(2 →)n and (→ 6)-α-D-Glc-(1 → 4)-α-Neu5Ac-(2 →)n, respectively. These unusual heteropolymers represent unique examples of a conjugation between sialic acid and hexosyl-sugars in a polymer chain. Moreover, despite the various catalytic strategies needed for sialic acid and hexose transfer, single enzymes (SiaDW-135/Y) have been identified to form these heteropolymers. Here we used SiaDW-135 as a model system to delineate structure-function relationships. In size exclusion chromatography active SiaDW-135 migrated as a monomer. Fold recognition programs suggested two separate glycosyltransferase domains, both containing a GT-B-fold. Based on conserved motifs predicted folds could be classified as a hexosyl- and sialyltransferase. To analyze enzyme properties and interplay of the two identified glycosyltransferase domains, saturation transfer difference NMR and mutational studies were carried out. Simultaneous and independent binding of UDP-Gal and CMP-Sia was seen in the absence of an acceptor as well as when the catalytic cycle was allowed to proceed. Enzyme variants with only one functionality were generated by site-directed mutagenesis and shown to complement each other in trans when combined in an in vitro test system. Together the data strongly suggests that SiaDW-135 has evolved by fusion of two independent ancestral genes encoding sialyl- and galactosyltransferase activity.

@article{romanow_biochemical_2013, abstract = {Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. Crucial virulence determinants of pathogenic Nm strains are the polysaccharide capsules that support invasion by hindering complement attack. In {NmW-135} and {NmY} the capsules are built from the repeating units (→ 6)-α-D-Gal-(1 → 4)-α-{Neu5Ac-(2} →)n and (→ 6)-α-D-Glc-(1 → 4)-α-{Neu5Ac-(2} →)n, respectively. These unusual heteropolymers represent unique examples of a conjugation between sialic acid and hexosyl-sugars in a polymer chain. Moreover, despite the various catalytic strategies needed for sialic acid and hexose transfer, single enzymes ({SiaDW-135/Y)} have been identified to form these heteropolymers. Here we used {SiaDW-135} as a model system to delineate structure-function relationships. In size exclusion chromatography active {SiaDW-135} migrated as a monomer. Fold recognition programs suggested two separate glycosyltransferase domains, both containing a {GT-B-fold.} Based on conserved motifs predicted folds could be classified as a hexosyl- and sialyltransferase. To analyze enzyme properties and interplay of the two identified glycosyltransferase domains, saturation transfer difference {NMR} and mutational studies were carried out. Simultaneous and independent binding of {UDP-Gal} and {CMP-Sia} was seen in the absence of an acceptor as well as when the catalytic cycle was allowed to proceed. Enzyme variants with only one functionality were generated by site-directed mutagenesis and shown to complement each other in trans when combined in an in vitro test system. Together the data strongly suggests that {SiaDW-135} has evolved by fusion of two independent ancestral genes encoding sialyl- and galactosyltransferase activity.}, added-at = {2013-10-04T09:46:01.000+0200}, author = {Romanow, Angela and Haselhorst, Thomas and Stummeyer, Katharina and Claus, Heike and Bethe, Andrea and Mühlenhoff, Martina and Vogel, Ulrich and von Itzstein, Mark and Gerardy-Schahn, Rita}, biburl = {https://www.bibsonomy.org/bibtex/24e2453f0abdb45e274e80841fee20842/ag_vogel}, doi = {10.1074/jbc.M113.452276}, interhash = {30ac1222bb5d09ec094c828607bf35bb}, intrahash = {4e2453f0abdb45e274e80841fee20842}, issn = {1083-{351X}}, journal = {The Journal of biological chemistry}, keywords = {Bacterial Bacterial_Capsules Bacterial_Proteins Evolution Gene_Fusion Humans Meningitis Meningococcal Molecular Mutagenesis Neisseria_meningitidis Polysaccharides Protein_Structure Sepsis Serogroup_W-135 Sialyltransferases Site-Directed Tertiary Uridine_Diphosphate_Galactose bacterial_capsules}, language = {eng}, month = apr, note = {{PMID:} 23439648}, number = 17, pages = {11718--11730}, timestamp = {2013-10-04T09:46:01.000+0200}, title = {Biochemical and biophysical characterization of the sialyl-/hexosyltransferase synthesizing the meningococcal serogroup W135 heteropolysaccharide capsule}, volume = 288, year = 2013 }