PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal: CRYSTAL STRUCTURE, INHIBITION, AND REACTION MECHANISM.

Autor:	Drees SL; From the Institute for Molecular Microbiology and Biotechnology and., Li C; Schools of Pharmacy and., Prasetya F; Schools of Pharmacy and., Saleem M; Schools of Pharmacy and., Dreveny I; Schools of Pharmacy and., Williams P; Life Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom., Hennecke U; Organic Chemistry Institute, University of Münster, D-48149 Münster, Germany and the Centre for Biomolecular Sciences., Emsley J; Schools of Pharmacy and jonas.emsley@nottingham.ac.uk., Fetzner S; From the Institute for Molecular Microbiology and Biotechnology and fetzner@uni-muenster.de.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2016 Mar 25; Vol. 291 (13), pp. 6610-24. Date of Electronic Publication: 2016 Jan 25.
DOI:	10.1074/jbc.M115.708453
Abstrakt:	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. (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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