PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism
Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyz...
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| Format: | Article |
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American Society for Biochemistry and Molecular Biology
2016
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| Online Access: | https://eprints.nottingham.ac.uk/50477/ |
| _version_ | 1848798260463927296 |
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| author | Drees, Steffen Lorenz Li, Chan Prasetya, Fajar Saleem, Muhammad Dreveny, Ingrid Williams, Paul Hennecke, Ulrich Emsley, Jonas Fetzner, Susanne |
| author_facet | Drees, Steffen Lorenz Li, Chan Prasetya, Fajar Saleem, Muhammad Dreveny, Ingrid Williams, Paul Hennecke, Ulrich Emsley, Jonas Fetzner, Susanne |
| author_sort | Drees, Steffen Lorenz |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis. |
| first_indexed | 2025-11-14T20:16:57Z |
| format | Article |
| id | nottingham-50477 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:16:57Z |
| publishDate | 2016 |
| publisher | American Society for Biochemistry and Molecular Biology |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-504772020-05-04T17:40:33Z https://eprints.nottingham.ac.uk/50477/ PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism Drees, Steffen Lorenz Li, Chan Prasetya, Fajar Saleem, Muhammad Dreveny, Ingrid Williams, Paul Hennecke, Ulrich Emsley, Jonas Fetzner, Susanne Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis. American Society for Biochemistry and Molecular Biology 2016-03-25 Article PeerReviewed Drees, Steffen Lorenz, Li, Chan, Prasetya, Fajar, Saleem, Muhammad, Dreveny, Ingrid, Williams, Paul, Hennecke, Ulrich, Emsley, Jonas and Fetzner, Susanne (2016) PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism. Journal of Biological Chemistry, 291 (13). pp. 6610-6624. ISSN 0021-9258 http://www.jbc.org/content/291/13/6610 doi:10.1074/jbc.M115.708453 doi:10.1074/jbc.M115.708453 |
| spellingShingle | Drees, Steffen Lorenz Li, Chan Prasetya, Fajar Saleem, Muhammad Dreveny, Ingrid Williams, Paul Hennecke, Ulrich Emsley, Jonas Fetzner, Susanne PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| title | PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| title_full | PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| title_fullStr | PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| title_full_unstemmed | PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| title_short | PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| title_sort | pqsbc, a condensing enzyme in the biosynthesis of the pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism |
| url | https://eprints.nottingham.ac.uk/50477/ https://eprints.nottingham.ac.uk/50477/ https://eprints.nottingham.ac.uk/50477/ |