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 (â 6)-α-D-Gal-(1 â 4)-α-Neu5Ac-(2 â)n and (â 6)-α-D-Glc-(1 â 4)-α-Neu5Ac-(2 â)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.
Subject(s)
Bacterial Capsules/metabolism , Bacterial Proteins/metabolism , Evolution, Molecular , Neisseria meningitidis, Serogroup W-135/enzymology , Polysaccharides, Bacterial/biosynthesis , Sialyltransferases/metabolism , Bacterial Capsules/chemistry , Bacterial Capsules/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Gene Fusion/physiology , Humans , Meningitis, Meningococcal/enzymology , Meningitis, Meningococcal/genetics , Meningitis, Meningococcal/pathology , Mutagenesis, Site-Directed , Neisseria meningitidis, Serogroup W-135/genetics , Neisseria meningitidis, Serogroup W-135/pathogenicity , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/genetics , Protein Structure, Tertiary , Sepsis/enzymology , Sepsis/genetics , Sepsis/pathology , Sialyltransferases/chemistry , Sialyltransferases/genetics , Uridine Diphosphate Galactose/chemistry , Uridine Diphosphate Galactose/genetics , Uridine Diphosphate Galactose/metabolismABSTRACT
The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD(W-135) and SiaD(Y), differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD(W-135) and SiaD(Y) were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.
