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.
biosynthesis, Neisseria meningitidis, sialic acid, glycosyltransferase, capsule polymerases, NmW-135
NCBI PubMed ID: 23439648Publication DOI: 10.1074/jbc.M113.452276Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: gerardy-schahn.rita@mh-hannover.de
Institutions: From the Institute for Cellular Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
Methods: SDS-PAGE, DNA techniques, genetic methods, radioactivity measurement, STD NMR
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