Molecular mechanism of biofilm disruption by fungal and bacterial glycoside hydrolases.

During infection, the fungal pathogen forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated -acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3 and PelA, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelA was solved to 1.54 Å and structure alignment to Sph3 revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelA hydrolyzed partially deacetylated substrates better than Sph3, a finding that agrees well with PelA's highly electronegative binding cleft the neutral surface present in Sph3 Our insight into PelA's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.