The Aspergillus fumigatus sialidase is a KDNase: structural and mechanistic insights

Judith C Telford, Juliana H F Yeung, Guogang Xu, Milton J Kiefel, Andrew G Watts, Stefan Hader, Jefferson Chan, Andrew J Bennet, Margo M Moore, Garry L Taylor

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

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 to bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared to bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate due to 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 utilise KDN as a sole carbon source. A 1.4Angstrom 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 analogue 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related KDN2en that we show bound to the enzyme in a 1.84Angstrom resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5Angstrom 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
JournalJournal of Biological Chemistry
Volume286
Issue number12
Early online date19 Jan 2011
DOIs
Publication statusPublished - 2011

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