P. aeruginosa SGNH Hydrolase-Like Proteins AlgJ and AlgX Have Similar Topology but Separate and Distinct Roles in Alginate Acetylation

Abstract

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.

Biological function of a polysaccharide degrading enzyme in the periplasm

“…After crossing the IM, poly-M is transported through the periplasm, the space between the inner and outer membranes (OM), by a membrane spanning multi-protein machine (Alg44, X, G, K)141617 to the OM protein, AlgE, for secretion181920. In the periplasm, poly-M is modified by O-acetylation (AlgI, J, F and X)212223 and epimerization (AlgG)242526. These modifications add acetyl groups to the C-2 and/or C-3 positions of M residues2122 and convert M residues into G residues2426, respectively.…”

“…In the periplasm, poly-M is modified by O-acetylation (AlgI, J, F and X)212223 and epimerization (AlgG)242526. These modifications add acetyl groups to the C-2 and/or C-3 positions of M residues2122 and convert M residues into G residues2426, respectively. Both modifications affect polymer composition and its barrier properties against therapeutic intervention and host immunity14272829.…”

Genomics Reveals a Unique Clone of Burkholderia cenocepacia Harboring an Actively Excising Novel Genomic Island

“…This GEI has one gene that encodes for a protein having a conserved domain for SGNH hydrolase, which belongs to a diverse family of lipases and esterases. In Pseudomonas aeruginosa , SGNH hydrolases are involved in acetylation of alginate, which is a long sugar polymer and the main component of Pseudomonas biofilm (Baker et al, 2014). Acetylation of alginate during its biosynthesis makes bacterial biofilm less susceptible to antibiotics, disinfectants and enable them to evade the immune system of a host (Ertesvåg, 2015).…”

Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

“…In the periplasm, M residues can be epimerized to G by AlgG (12). They may also be acetylated selectively at O-2= and/or O-3= positions by AlgX (13). Additionally, AlgI, AlgJ, and AlgF are involved in alginate acetylation (14,15).…”

“…AlgK is a periplasmic lipoprotein bound to the outer membrane and is important, but not essential, for the localization of the porin AlgE. Figure 1 represents the proposed model for alginate biosynthesis machinery based on recent studies (13,21,22). The interactions between different Alg proteins, assumed to contribute to the formation of the mentioned protein scaffold, have been extensively studied by mutual stability and pulldown analyses.…”