Objectives: The aim of this study is investigate the role of NanOU in sialic acid transport.
Methods: The role of nanOU in sialic acid transport was examined by genetic transplantation into an E. coli sialic acid auxotroph, while recombinant NanU protein was expressed, purified and its biochemical, and structural properties investigated by gel filtration, tryptophan fluorescence titration, and X-ray crystallography. Cellular localisation was confirmed by immunoblotting.
Results: We showed that nanOU genes from both T. forsythia and the gastrointestinal bacterium Bacteroides fragilis complement the sialic acid growth defect of an E. coli sialic acid auxotroph and established dependence on an intact TonB-ExbB-ExbD complex. In contrast, nanO partially restored growth while nanU did not, indicating NanU is required for optimal function. Using anti-NanU antiserum, we determined that NanU is localised in the outer membrane. Furthermore, NanU binds sialic acid with a Kd of ~400 nM in addition to several other sialic acid analogues including the antiflu drug Zanamavir. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure that contains a novel motif characterised by an extended helix that might determine sugar-binding specificity.
Conclusion: These data structurally and biochemically characterise the first bacterial extracellular sialic acid binding protein and define a new class Polysaccharide Utilisation Loci (PUL) specific for sialic acid.