A Mechanistic Basis for Phosphoethanolamine Modification of the Cellulose Biofilm Matrix in .

Biofilms are communities of self-enmeshed bacteria in a matrix of exopolysaccharides. The widely distributed human pathogen and commensal produces a biofilm matrix composed of phosphoethanolamine (pEtN)-modified cellulose and amyloid protein fibers, termed curli. The addition of pEtN to the cellulose exopolysaccharide is accomplished by the action of the pEtN transferase, BcsG, and is essential for the overall integrity of the biofilm. Here, using the synthetic co-substrates nitrophenyl phosphoethanolamine and β-d-cellopentaose, we demonstrate using an pEtN transferase assay that full activity of the pEtN transferase domain of BcsG from (BcsG) requires Zn binding, a catalytic nucleophile/acid-base arrangement (Ser/Cys/His), disulfide bond formation, and other newly uncovered essential residues. We further confirm that BcsG catalysis proceeds by a ping-pong bisubstrate-biproduct reaction mechanism and displays inefficient kinetic behavior (/ = 1.81 × 10 ± 2.81 × 10 M s), which is typical of exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected by ), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes, which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family, including the intrinsic and mobile colistin resistance factors.