Abstact

Exopolysaccharides are key matrix determinants that provide structural integrity and regulate biomechanical properties of microbial biofilms. Biofilm exopolysaccharides often undergo modifications that determine their functional properties and localization. In B. cereus ATCC 10987, PelA(DA) expressed from the pelDEA(DA)FG operon is a putative deacetylase required for Pel-dependent biofilm formation. To understand the molecular basis of Pel deacetylation in B. cereus ATCC 10987, we determined the crystal structure of PelA(DA) to 2.51 A. PelA(DA) adopts a distinct three-domain arrangement. We demonstrate in vitro that PelA(DA) deacetylates alpha-1,4-linked GalNAc substrates in a length-dependent manner and that the N-terminal domain functions as a carbohydrate binding module (CBM) capable of binding both GalNAc and partially deacetylated oligosaccharides. We found that the CBM domain together with the carbohydrate esterase (CE) domain forms an elongated carbohydrate binding cleft and that each domain is the founding member of two new CAZy families, CBMxx and CExx, respectively. Further, in vivo mutagenesis demonstrated that the catalytic activity of PelA(DA) is required for Pel biosynthesis in B. cereus ATCC 10987. Employing AlphaFold, we propose a model wherein the N-terminal transmembrane helix of PelA(DA) interacts with PelG. This interaction positions the protein to accept the polymer for deacetylation as it emerges from the cytoplasmic membrane. The work presented herein offers insight into the role of PelA(DA) in Pel biosynthesis and modification in B. cereus ATCC 10987.