Abstract
The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition "pinch point" that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.
Highlights
AlgX. Residues Tyr-328 (AlgX) is required for the biosynthesis and export of the exopolysaccharide alginate
Our data suggest that AlgX plays two roles in alginate biosynthesis; does the protein protect the polymer as it passages through the periplasm [29], but it plays a role in its acetylation, an activity that is controlled by the Ser-His-Asp catalytic triad
AlgX contains two domains, an N-terminal SGNH hydrolase-like domain and a C-terminal carbohydrate-binding module. This combination is not unexpected because CBMs are commonly appended to enzymatic domains, including SGNH hydrolases, where they function to bring the relevant polysaccharide into close proximity to the active site [68, 79]
Summary
AlgX is required for the biosynthesis and export of the exopolysaccharide alginate. Results: The structure of AlgX has been determined, and the functional characterization of AlgX and mutant variants has been performed. Bioinformatics analyses suggest that AlgX is a two-domain protein, with an N-terminal region that is predicted to share structural homology with members of the SGNH hydrolase superfamily [34] This region of the protein shares ϳ69% similarity and 30% identity with AlgJ, the putative alginate acetyltransferase [24], and has been proposed to have a similar function [19]. Site-specific mutation in vivo of the Ser-His-Asp triad and other key residues in the putative active site results in either non-acetylated or acetylationreduced alginate polymer, respectively. Our data suggest that AlgX plays two roles in alginate biosynthesis; does the protein protect the polymer as it passages through the periplasm [29], but it plays a role in its acetylation, an activity that is controlled by the Ser-His-Asp catalytic triad. The structure of AlgX provides insight into the function of AlgJ and other polysaccharide acetyltransferases involved in the acetylation of biofilm polymers and other bacterial polysaccharides, such as cellulose and peptidoglycan, respectively
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