title: PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal: CRYSTAL STRUCTURE, INHIBITION, AND REACTION MECHANISM.
abstract: Pseudomonas aeruginosaproduces 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 forin vitrobinding 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.
authors.affiliation: From the Institute for Molecular Microbiology and Biotechnology and.
authors.forename: Steffen Lorenz
authors.initials: SL
authors.lastname: Drees
authors.affiliation: Schools of Pharmacy and.
authors.forename: Chan
authors.initials: C
authors.lastname: Li
authors.affiliation: Schools of Pharmacy and.
authors.forename: Fajar
authors.initials: F
authors.lastname: Prasetya
authors.affiliation: Schools of Pharmacy and.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Saleem
authors.affiliation: Schools of Pharmacy and.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Dreveny
authors.affiliation: Life Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Williams
authors.affiliation: Organic Chemistry Institute, University of Münster, D-48149 Münster, Germany and the Centre for Biomolecular Sciences.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hennecke
authors.affiliation: Schools of Pharmacy and jonas.emsley@nottingham.ac.uk.
authors.forename: Jonas
authors.initials: J
authors.lastname: Emsley
authors.affiliation: From the Institute for Molecular Microbiology and Biotechnology and fetzner@uni-muenster.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fetzner
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D042462:4-Quinolones / Q000737:chemistry* / Q000378:metabolism; D000098:Acetophenones / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D062365:Aminobenzoates / Q000737:chemistry* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C528166:2-heptyl-4-quinolone; D042462:4-Quinolones; D000098:Acetophenones; D000214:Acyl Coenzyme A; D062365:Aminobenzoates; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008024:Ligands; D021122:Protein Subunits; D011994:Recombinant Proteins; C034681:octanoyl-coenzyme A; C055495:2-aminoacetophenone; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase
keywords: 2-alkyl-4(1H)-quinolones; FabH; Pseudomonas aeruginosa (P. aeruginosa); Pseudomonas quinolone signal; biosynthesis; condensing enzyme; crystal structure; quorum sensing; secondary metabolism
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