The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

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-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.

FIGURE 1.

Chemical structures of ligands used in this study.

FIGURE 2.

Overall structure of AfS. Top panel, cartoon drawn from blue at the N terminus to red at the C terminus. The black sphere below the cap domain and above the active site is a metal-binding site. Bottom panel, a surface representation in the same orientations as the images above, colored according to electrostatic potential from −7KT to +7KT, calculated using APBS (50). This and other figures were drawn using PyMol (51). The locations of KDN-binding sites observed in the crystal structures are indicated.

FIGURE 3.

Comparison of the active sites of AfS and MvS. Stereo image showing a superposition of apo-AfS (green carbons) with a Neu5Ac2en complex of MvS (gray carbons) (Protein Data Bank code 1eus). Key conserved residues are labeled in the order AfS/MvS, and Arg¹⁷¹ of AfS is highlighted.

FIGURE 4.

KDN recognition by AfS. A, stereo image of a F_o − F_c electron density map, contoured at 3 σ. B, stereo image of the KDN-binding sites showing hydrogen bond interactions (≤3.2 Å) drawn as dotted lines.

FIGURE 5.

KDN bound in the active site (left) and second binding site, with the bound glycerol also shown.

FIGURE 6.

Complex of AfS with KDN2en. Stereo image of a F_o − F_c electron density map contoured at 2.5 σ around KDN2en. Direct hydrogen bond interactions between the ligand and protein are shown.

FIGURE 7.

Covalent intermediate. A, stereo drawing showing the 2F_o − F_c electron density map (contoured at 1 σ) displayed around the 3-F-β-KDN covalently bound to Tyr³⁵⁸. B, superposition of the complexes of AfS with KDN (green carbons), KDN2en (yellow carbons), and 3-F-β-KDN (magenta carbons). All of the protein carbon atoms are drawn in gray. The conserved interactions between the arginine triad and the carboxylate of the ligands are drawn as dotted lines.

FIGURE 8.

The ¹H NMR spectrum showing hydration of KDN-MU by AfS. The bottom spectrum is that of KDN-MU, and the upper spectra are taken at the time intervals shown after the addition of AfS.

FIGURE 9.

Growth of A. fumigatus on different carbon sources. K indicates the defined medium (Kafer's) with the additional carbon sources: Glu, glucose; Man, mannose; SA, Neu5Ac. All of the carbon sources were 25 mm except for K (+0.05 Man), which contained 1.25 mm mannose.