The Structure of Clostridium perfringens NanI Sialidase and Its Catalytic Intermediates

Abstract

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97 Å resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97 Å resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5 Å resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2 Å resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested.

The Aspergillus fumigatus Sialidase Is a 3-Deoxy-d-glycero-d-galacto-2-nonulosonic Acid Hydrolase (KDNase)

“…7B). With a KDN glycan it is likely that catalysis would proceed with the concomitant release of the aglycon moiety as described for other sialidases (13). This similarity between the mechanism of AfS and other sialidases is further supported by the NMR data (Fig.…”

“…2). This is in contrast to secreted bacterial sialidases that tend to have a highly acidic surface on one side that, it is suggested, helps orient the enzyme in the neighborhood of a negatively charged surface (13,15,26). Behind the active site and below the cap domain there is a metal-binding site (Fig.…”

“…2). A more extensive excursion at the same topological position exists in the NanI sialidase from C. perfringens (13) and the NanA/NanB sialidases from S. pneumoniae (15,16). AfS is a basic protein with a theoretical pI of 9.3, and this is reflected in the electrostatic potential surface that shows a predominantly positive surface around the active site and extending in a groove down and below the molecule (Fig.…”

Neuraminidase-1, a Subunit of the Cell Surface Elastin Receptor, Desialylates and Functionally Inactivates Adjacent Receptors Interacting with the Mitogenic Growth Factors PDGF-BB and IGF-2

“…Importantly, in subconfluent cultures of AoSMCs maintained in serum-free media, pretreatment with C. perfringens Nase, which like mammalian Neu1 exclusively removes sialic acid residues from monovalent sialylated glycoproteins, 51 completely abolished the response of the AoSMCs to recombinant PDGF-BB or IGF-2, but not diminish the mitogenic effect of IL-1␤, which receptors are not sialylated glycoprotein's. 68,69 Thus, the exogenous C. perfringens neuraminidase altered cellular responsiveness to PDGF-BB or IGF-2, probably mimicking the overexpression of cell surface Neu1.…”

“…[51][52][53][54] This particular bacterial neuraminidase was chosen because it (like human Neu1) preferentially removes terminal sialic acids from monovalent sialylated glycoproteins but not from sialylated gangliosides, as other bacterial sialidases do. We anticipated that the biological effects observed after treatment of cultured SMCs with the C. perfringens neuraminidase would differ from the effects recorded after treatment with Vibrio cholerae neuraminidase, [55][56][57] which preferentially hydrolyzes both Ϫ2,3-and Ϫ2,6-linked sialic acid residues from higher-order gangliosides.…”