Abstract
Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4-linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (β/α)8-fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the -2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi.
Highlights
Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections
The extracellular region was predicted by BlastP and the dbCAN2 annotation server to include a glycoside hydrolase family 114 (GH114) domain between residues 83 and 314 [29, 30]
The location of ega3 within the GAG cluster and its up-regulation during biofilm formation suggest that the GH114 domain encoded by this gene likely plays a role in the GAG biosynthesis
Summary
To gain insight into the structure and function of Ega, we first examined its amino acid sequence to determine its domain structure and identify boundaries that could be used for construct design. The TMHMM server [28] predicted that the N-terminal region of the protein contains a putative transmembrane helix between residues 22 and 45, with the N terminus residing in the cytosol (Fig. 1A). Asn-222 was given a low score by the server, and Asn-253 was not predicted as the sequon was Asn-Xaa-Cys instead of Asn-Xaa-(Ser/Thr) [32] Whether any of these sites are glycosylated in the native protein has yet to be determined, but it is interesting to note that only the sequons at Asn-92 and Asn-222 are conserved within Ega orthologues. In addition to T. maritima TM1410, which was used for phase determination, the structural similarity server, DALI [33], found that Ega is similar to the hydrolase domain of Pseudomonas aeruginosa PelA (PelAh, PDB 5TCB). Data collection Beamline Wavelength (Å) Space group Unit-cell parameters (Å,°) Resolution (Å) Total no. of reflections No of unique reflections Redundancy Completeness (%) Average I/(I) Rmerge (%)a CC1⁄2 (%)b
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