Background: The Streptococcus pneumoniae sialidase NanC produces a nonspecific inhibitor of hydrolytic sialidases. Results: The NanC crystal structure is presented in complex with mechanistically relevant ligands. Conclusion: A constricted and hydrophobic active site produces 2-deoxy-2,3-didehydro- N -acetylneuraminic acid (Neu5Ac2en, also known as DANA) via a covalent intermediate and direct proton abstraction by a catalytic aspartic acid. Significance: Insights into an unusual reaction mechanism will aid the design of sialidase inhibitors. Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro- N -acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β- N -acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N -acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.
【저자키워드】 sialic acid, pneumococcus, Streptococcus, Carbohydrate-binding module, neuraminidase, sialidase, glycoside hydrolase, NanC, neu5ac2en,