The Aspergillus fumigatus Sialidase Is a 3-Deoxy-d-glycero-d-galacto-2-nonulosonic Acid Hydrolase (KDNase)

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.

The Aspergillus fumigatus Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice

“…Compared to Kdn, Neu5Ac (labeled sialic acid in Figure 1 ) is a poor carbon source for A. fumigatus ; however, the Δ kdnase strain had even lower growth than WT on Neu5Ac. These data also confirm our previous finding that Kdn is strongly preferred to Neu5Ac in A. fumigatus ( Telford et al, 2011 ).…”

“…In contrast, we previously showed that Neu5Ac2en did not inhibit the recombinant A. fumigatus Kdnase even at concentrations up to 10 mM, but was inhibited by the related Kdn analog, 2,3-didehydro-2,3-dideoxy- D - glycero - D - galacto -non-2-ulosonic acid (Kdn2en). Furthermore, Kdn2en bound to the enzyme in a 1.84 Å-resolution crystal structure ( Telford et al, 2011 ). Thus, it is possible to selectively inhibit Kdnase without affecting host sialidases.…”

“…Growth in medium containing human serum induced sialidase gene expression ( Warwas et al, 2010 ). Subsequent studies revealed that the A. fumigatus sialidase activity was not inhibited by the classical sialidase inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) ( Telford et al, 2011 ). Furthermore, the crystallized A. fumigatus sialidase covalently bound 3-F-β-Kdn in the enzyme active site.…”

“…Furthermore, the crystallized A. fumigatus sialidase covalently bound 3-F-β-Kdn in the enzyme active site. Kinetic analysis showed that the catalytic efficiency ( k cat /K m ) was 5 orders of magnitude higher with Kdn-methylumbelliferone compared to the Neu5Ac substrate ( Telford et al, 2011 ). Hence, the A. fumigatus sialidase is a Kdnase but its importance to survival of A. fumigatus in vitro and in vivo is unknown.…”

Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens

“…Because the half-life for mutarotation of Neu5Ac at pD 6.7 is around 25 min (17), and that of Kdn appears to be similar (18), it would seem likely that any hydrated intermediate released into solution by UGL would accumulate under the experimental conditions used, and thus become detectable by 1 HNMR, before formation of the final product. These results were interpreted as suggesting that rearrangement of the hydrated intermediate is indeed catalyzed within the UGL active site, as assumed previously.…”