The Aspergillus fumigatus Sialidase Is a 3-Deoxy-D-glycero-D-galacto-2-nonulosonic Acid Hydrolase (KDNase): Structural and mechanistic insights

J C Telford, J H F Yeung, G G Xu, M J Kiefel, Andrew Watts, Stefan Hader, J Chan, A J Bennet, M M Moore, G L Taylor

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Abstract

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-angstrom resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3dideoxy-D-glycero-D-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-angstrom resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-angstrom resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.
Original languageEnglish
Pages (from-to)10783-10792
Number of pages10
JournalJournal of Biological Chemistry
Volume286
Issue number12
DOIs
Publication statusPublished - 25 Mar 2011

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    Telford, J. C., Yeung, J. H. F., Xu, G. G., Kiefel, M. J., Watts, A., Hader, S., Chan, J., Bennet, A. J., Moore, M. M., & Taylor, G. L. (2011). The Aspergillus fumigatus Sialidase Is a 3-Deoxy-D-glycero-D-galacto-2-nonulosonic Acid Hydrolase (KDNase): Structural and mechanistic insights. Journal of Biological Chemistry, 286(12), 10783-10792. https://doi.org/10.1074/jbc.M110.207043