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. 2008 Apr 4;283(14):9080-8.
doi: 10.1074/jbc.M710247200. Epub 2008 Jan 24.

The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates

Simon L Newstead  1 , Jane A Potter, Jennifer C Wilson, Guogang Xu, Chin-Hsiang Chien, Andrew G Watts, Stephen G Withers, Garry L Taylor
Affiliations

The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates

Simon L Newstead et al. J Biol Chem. .

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 0.97A 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.

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Figures

FIGURE 1.
FIGURE 1.
Ligands used in this study. Structure 1, Neu5Ac; structure 2, Neu5Ac2en; and structure 3, 2,3-difluoro-N-acetylneuramic acid (2,3F-Neu5Ac).
FIGURE 2.
FIGURE 2.
Overall structure of the NanI sialidase. A and B represent orthogonal views of a comparison of the fold of NanI (yellow) with the leech sialidase (blue). Sialic acid is drawn as spheres to locate the active site. C and D show a surface representation of NanI, in the same orientations as above, colored according to electrostatic potential from -7 kT/e to +7 kT/e, calculated using APBS (26).
FIGURE 3.
FIGURE 3.
Stereo images of unbiased Fo - Fc electron density maps contoured at 3σ with the final refined coordinates superimposed. A, Neu5Ac. B, Neu5Ac2en. C, 3-fluoro-2-deoxy-N-acetylneuraminic acid covalently linked to Tyr655.
FIGURE 4.
FIGURE 4.
Stereo views of the NanI active site. A, complex with Neu5Ac showing the hydrogen-bonding interactions as green dotted lines. B, superposition of the three ligand complexes and ligand free structure: Neu5Ac with yellow carbons, Neu5Ac2en with magenta carbons, and the covalent fluorinated ligand with cyan carbons.
FIGURE 5.
FIGURE 5.
The 1H NMR spectrum (600 MHz, 285 K) of Neu5Ac2en in the presence of NanI 0 h after preparation of the sample (A), and the same sample after 10 days (B).
FIGURE 6.
FIGURE 6.
Proposed mechanism for the hydration of Neu5Ac2en to Neu5Ac.
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