P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.
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 cons...
| Main Authors: | , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-08-01
|
| Series: | PLoS Pathogens |
| Online Access: | http://europepmc.org/articles/PMC4148444?pdf=render |
| _version_ | 1819074032033595392 |
|---|---|
| author | Perrin Baker Tyler Ricer Patrick J Moynihan Elena N Kitova Marthe T C Walvoort Dustin J Little John C Whitney Karen Dawson Joel T Weadge Howard Robinson Dennis E Ohman Jeroen D C Codée John S Klassen Anthony J Clarke P Lynne Howell |
| author_facet | Perrin Baker Tyler Ricer Patrick J Moynihan Elena N Kitova Marthe T C Walvoort Dustin J Little John C Whitney Karen Dawson Joel T Weadge Howard Robinson Dennis E Ohman Jeroen D C Codée John S Klassen Anthony J Clarke P Lynne Howell |
| author_sort | Perrin Baker |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T18:03:03Z |
| format | Article |
| id | doaj.art-e34e3f18f1c64d8d892971bdb09e1da6 |
| institution | Directory Open Access Journal |
| issn | 1553-7366 1553-7374 |
| language | English |
| last_indexed | 2024-12-21T18:03:03Z |
| publishDate | 2014-08-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj.art-e34e3f18f1c64d8d892971bdb09e1da62022-12-21T18:55:01ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742014-08-01108e100433410.1371/journal.ppat.1004334P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.Perrin BakerTyler RicerPatrick J MoynihanElena N KitovaMarthe T C WalvoortDustin J LittleJohn C WhitneyKaren DawsonJoel T WeadgeHoward RobinsonDennis E OhmanJeroen D C CodéeJohn S KlassenAnthony J ClarkeP Lynne HowellThe 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.http://europepmc.org/articles/PMC4148444?pdf=render |
| spellingShingle | Perrin Baker Tyler Ricer Patrick J Moynihan Elena N Kitova Marthe T C Walvoort Dustin J Little John C Whitney Karen Dawson Joel T Weadge Howard Robinson Dennis E Ohman Jeroen D C Codée John S Klassen Anthony J Clarke P Lynne Howell P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. PLoS Pathogens |
| title | P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. |
| title_full | P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. |
| title_fullStr | P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. |
| title_full_unstemmed | P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. |
| title_short | P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation. |
| title_sort | p aeruginosa sgnh hydrolase like proteins algj and algx have similar topology but separate and distinct roles in alginate acetylation |
| url | http://europepmc.org/articles/PMC4148444?pdf=render |
| work_keys_str_mv | AT perrinbaker paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT tylerricer paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT patrickjmoynihan paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT elenankitova paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT marthetcwalvoort paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT dustinjlittle paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT johncwhitney paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT karendawson paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT joeltweadge paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT howardrobinson paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT denniseohman paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT jeroendccodee paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT johnsklassen paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT anthonyjclarke paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation AT plynnehowell paeruginosasgnhhydrolaselikeproteinsalgjandalgxhavesimilartopologybutseparateanddistinctrolesinalginateacetylation |