title: The Crystal Structure of Burkholderia cenocepacia DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis.
abstract: Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Spadaro
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Viola C
authors.initials: VC
authors.lastname: Scoffone
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Laurent R
authors.initials: LR
authors.lastname: Chiarelli
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fumagalli
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Buroni
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Riccardi
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia , Via Ferrata 9/A, 27100 Pavia, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Forneris
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D057508:Burkholderia cenocepacia / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D011487:Protein Conformation; D053038:Quorum Sensing; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids