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

Drees, Steffen Lorenz and Li, Chan and Prasetya, Fajar and Saleem, Muhammad and Dreveny, Ingrid and Williams, Paul and Hennecke, Ulrich and 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

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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 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.

Item Type: Article
Schools/Departments: University of Nottingham, UK > Faculty of Medicine and Health Sciences > School of Life Sciences
University of Nottingham, UK > Faculty of Science > School of Pharmacy
Identification Number: 10.1074/jbc.M115.708453
Depositing User: Eprints, Support
Date Deposited: 16 Mar 2018 12:14
Last Modified: 17 Mar 2018 15:48
URI: http://eprints.nottingham.ac.uk/id/eprint/50477

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