The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.

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 0.97A 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 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A 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.

Galactose recognition by the carbohydrate-binding module of a bacterial sialidase.

Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catalytic domains, which help target the enzymes to appropriate substrates and thereby increase their catalytic efficiency. Sialidases hydrolyse the release of sialic acid from a variety of glycoconjugates and play significant roles in the pathogenesis of a number of important diseases. The sialidase from Micromonospora viridifaciens has a CBM which recognizes galactose. The CBM is linked to the catalytic domain by an immunoglobulin-like domain, resulting in the galactose binding site sitting above the catalytic site, suggesting an interplay between the two sites. By studying nine crystallographically independent structures of the M. viridifaciens sialidase, the relative flexibility of the three domains was analysed. A detailed study is also presented of the recognition of galactose and lactose by the M. viridifaciens CBM. The striking structure of this sialidase suggests a role for the CBM in binding to galactose residues unmasked by the adjacent catalytic site.