The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights.
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-Å 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-2,3-dideoxy-D-glycero-D-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å 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.
Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, Fife KY16 9ST, United Kingdom.
Judith C
JC
Telford
Juliana H F
JH
Yeung
Guogang
G
Xu
Milton J
MJ
Kiefel
Andrew G
AG
Watts
Stefan
S
Hader
Jefferson
J
Chan
Andrew J
AJ
Bennet
Margo M
MM
Moore
Garry L
GL
Taylor
D001232:Aspergillus fumigatus / Q000201:enzymology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D009439:Neuraminidase / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C091615:deaminoneuraminate hydrolase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid
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