Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6- N -acetyl- d -glucosamine

Abstract

Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de-N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N-and C-terminal domains forms a cleft required for the binding and de-N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB 310-672 ) directly binds PNAG oligomers with dissociation constants of ∼1-3 mM, and the structures of PgaB 310-672 in complex with β-1,6-(GlcNAc) 6 , GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de-N-acetylated PNAG (dPNAG). Furthermore, PgaB 310-672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB 42-655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB 310-672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.exopolysaccharide biosynthesis | glycobiology | carbohydrate binding | deacetylase

The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile

“…Tyr645, which is found at the C‐terminus of β8, was also identified in PgaB as being important to carbohydrate binding 26. β‐mannanase 4a was noted as possessing a cis‐peptide bond between the equivalent residue, Trp360, and Glu361 that was deduced to be important for the formation of the S 1 pocket 28.…”

Periplasmic de-acylase helps bacteria don their biofilm coat

“…Similarly, chitin can be converted to a partially de-N-acetylated form known as chitosan. In PNAS, Little et al (5) use structural, computational, and functional studies to explore the role of PgaB, a de-N-acetylase enzyme that is known to be critical in both the tailoring and extracellular export of the dPNAG component of biofilms produced by Escherichia coli.…”

“…Upon the basis of the PgaB structure, Little et al (5) have performed a series of functional, structural, and computational studies to comprehensively determine the roles of the individual domains of PgaB within the functional context of the whole enzyme and its role in dPNAG production and export. Enzyme assays show that the two-domain protein must be intact to catalyze de-N-acetylation of β-1,6-GlcNAc oligomers, neither single domain being active, either individually or in combination.…”

“…ABC-transporterdependent pathways for capsular polysaccharide export involve a multiprotein complex that spans the periplasm and funnels the polysaccharide out (12). In the case of synthase-dependent pathways, and dPNAG production and export in E. coli in particular, Little et al (5) show that PgaB is important not only in modifying the polysaccharide to its partially de-N-acetylated form in the periplasm, but also in closely interacting with the polymer, which is apparently wound about the C-terminal domain following its modification, moving processively over the surface of the protein before it is threaded through the β-barrel porin of the PgaA protein for transport across the outer membrane. These results suggest that such protein-polysaccharide interactions may be generally important for protecting or directing the polysaccharide in the periplasm during export by the synthase-dependent pathways widely involved in biofilm formation.…”

“…For example, a recent crystal structure of the BscA-BscB complex, which makes up the cellulose synthase in Rhodobacter sphaeroides (involved in biofilm formation in that organism), shows that the BscB extends 60 Å into the periplasm and includes two putative carbohydrate-binding domains that could interact closely with the nascent cellulose in that pathway and assist in directing its export (13). Little et al (5) present data suggesting that PgaB plays this role in dPNAG production in E. coli, in addition to tailoring the PNAG precursor to form the mature dPNAG polysaccharide. Evidently, PgaB is closely intertwined with dPNAG production in biofilm formation.…”