title: A Comparative Analysis of Acyl-Homoserine Lactone Synthase Assays.
abstract: Quorum sensing is cell-to-cell communication that allows bacteria to coordinate attacks on their hosts by inducing virulent gene expression, biofilm production, and other cellular functions, including antibiotic resistance. AHL synthase enzymes synthesize N-acyl-l-homoserine lactones, commonly referred to as autoinducers, to facilitate quorum sensing in Gram-negative bacteria. Studying the synthases, however, has proven to be a difficult road. Two assays, including a radiolabeled assay and a colorimetric (DCPIP) assay are well-documented in literature to study AHL synthases. In this paper, we describe additional methods that include an HPLC-based, C-S bond cleavage and coupled assays to investigate this class of enzymes. In addition, we compare and contrast each assay for both acyl-CoA- and acyl-ACP-utilizing synthases. The expanded toolkit described in this study should facilitate mechanistic studies on quorum sensing signal synthases and expedite discovery of antivirulent compounds.
authors.affiliation: Department of Chemistry and Biochemistry, Boise State University, 1910 University Drive, Boise, ID, 83725, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Shin
authors.affiliation: Department of Chemistry and Biochemistry, Boise State University, 1910 University Drive, Boise, ID, 83725, USA.
authors.forename: Nicole D
authors.initials: ND
authors.lastname: Frane
authors.affiliation: Department of Chemistry and Biochemistry, Boise State University, 1910 University Drive, Boise, ID, 83725, USA.|Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, PO Box 208114, New Haven, CT, 06520-8114, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Brecht
authors.affiliation: Department of Chemistry, Northwest Nazarene University, Nampa, ID, 83686, USA.|Loma Linda School of Medicine, Coleman Pavilion, 11175 Campus Street, Loma Linda, CA, 92350, USA.
authors.forename: Jesse
authors.initials: J
authors.lastname: Keeler
authors.affiliation: Department of Chemistry and Biochemistry, Boise State University, 1910 University Drive, Boise, ID, 83725, USA. rajnagarajan@boisestate.edu.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Nagarajan
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays; D006090:Gram-Negative Bacteria / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000032:analysis*; D013053:Spectrophotometry; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014969:Xanthine Oxidase; D008025:Ligases; C449816:N-acylhomoserine lactone synthase
keywords: acyl-homoserine lactone synthase; enzyme assays; methylthioadenosine nucleosidase; quorum sensing; xanthine oxidase