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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

Authors

Steffen Lorenz Drees

Chan Li

Fajar Prasetya

Muhammad Saleem

PAUL WILLIAMS paul.williams@nottingham.ac.uk
Professor of Molecular Microbiology

Ulrich Hennecke

prof JONAS EMSLEY jonas.emsley@nottingham.ac.uk
Professor of Macromolecularcrystallography

Susanne Fetzner



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.

Citation

Drees, S. L., Li, C., Prasetya, F., Saleem, M., Dreveny, I., Williams, P., …Fetzner, S. (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), https://doi.org/10.1074/jbc.M115.708453

Journal Article Type Article
Acceptance Date Jan 11, 2016
Online Publication Date Jan 25, 2016
Publication Date Mar 25, 2016
Deposit Date Mar 16, 2018
Publicly Available Date Mar 16, 2018
Journal Journal of Biological Chemistry
Electronic ISSN 0021-9258
Publisher American Society for Biochemistry and Molecular Biology
Peer Reviewed Peer Reviewed
Volume 291
Issue 13
DOI https://doi.org/10.1074/jbc.M115.708453
Public URL http://eprints.nottingham.ac.uk/id/eprint/50477
Publisher URL http://www.jbc.org/content/291/13/6610
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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