Steffen Lorenz Drees
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
CHAN LI chan.li@nottingham.ac.uk
Research Fellow
Fajar Prasetya
Muhammad Saleem
INGRID DREVENY ingrid.dreveny@nottingham.ac.uk
Associate Professor
PAUL WILLIAMS PAUL.WILLIAMS@NOTTINGHAM.AC.UK
Professor of Molecular Microbiology
Ulrich Hennecke
prof JONAS EMSLEY jonas.emsley@nottingham.ac.uk
Professor of Macromolecular Crystallography
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 | https://nottingham-repository.worktribe.com/output/779123 |
Publisher URL | http://www.jbc.org/content/291/13/6610 |
Contract Date | Mar 16, 2018 |
Files
J. Biol. Chem.-2016-Drees-6610-24.pdf
(4.6 Mb)
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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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