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PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism

DOI: 10.1074/jbc.M115.708453 DOI Help
PMID: 26811339 PMID Help

Authors: Steffen Lorenz Drees (University of Muenster) , Chan Li (University of Nottingham) , Fajar Prasetya (University of Nottingham) , Muhammad Saleem (University of Nottingham) , Ingrid Dreveny (University of Nottingham) , Paul Williams (University of Nottingham) , Ulrich Hennecke (University of Muenster) , Jonas Emsley (University of Nottingham) , Susanne Fetzner (University of Muenster)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: January 2016
Diamond Proposal Number(s): 10369

Abstract: 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 2Å resolution, revealing that PqsB and PqsC have a pseudo 2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site comprised of Cys129 and His269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes which 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.

Journal Keywords: biosynthesis; crystal structure; Pseudomonas aeruginosa (P. aeruginosa); quorum sensing; secondary metabolism; 2-alkyl-4(1H)-quinolone;s FabH; Pseudomonas quinolone signal; condensing enzyme

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography

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