title: Biochemical and biophysical characterization of the sialyl-/hexosyltransferase synthesizing the meningococcal serogroup W135 heteropolysaccharide capsule.
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.
authors: Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors: Angela
authors: A
authors: Romanow
authors: Thomas
authors: T
authors: Haselhorst
authors: Katharina
authors: K
authors: Stummeyer
authors: Heike
authors: H
authors: Claus
authors: Andrea
authors: A
authors: Bethe
authors: Martina
authors: M
authors: Mühlenhoff
authors: Ulrich
authors: U
authors: Vogel
authors: Mark
authors: M
authors: von Itzstein
authors: Rita
authors: R
authors: Gerardy-Schahn
mesh_terms: D016667:Bacterial Capsules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D050939:Gene Fusion / Q000502:physiology; D006801:Humans; D008585:Meningitis, Meningococcal / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D038582:Neisseria meningitidis, Serogroup W-135 / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D018805:Sepsis / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014531:Uridine Diphosphate Galactose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D014531:Uridine Diphosphate Galactose; D012799:Sialyltransferases