title: Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay for N-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes.
abstract: Quorum sensing, the bacterial cell-cell communication by small molecules, controls important processes such as infection and biofilm formation. Therefore, it is a promising target with several therapeutic and technical applications besides its significant ecological relevance. Enzymes inactivating N-acyl-l-homoserine lactones, the most common class of communication molecules among Gram-negative proteobacteria, mainly belong to the groups of quorum-quenching lactonases or quorum-quenching acylases. However, identification, characterization, and optimization of these valuable biocatalysts are based on a very limited number of fundamentally different methods with their respective strengths and weaknesses. Here, a (bio)chemical activity assay is described, which perfectly complements the other methods in this field. It enables continuous and high-throughput activity measurements of purified and unpurified quorum-quenching enzymes within several minutes. For this, the reaction products released by quorum-quenching lactonases and quorum-quenching acylases are converted either by a secondary enzyme or by autohydrolysis to l-homoserine. In turn, l-homoserine is detected by the previously described calcein assay, which is sensitive to α-amino acids with free N and C termini. Besides its establishment, the method was applied to the characterization of three previously undescribed quorum-quenching lactonases and variants thereof and to the identification of quorum-quenching acylase-expressing Escherichia coli clones in an artificial library. Furthermore, this study indicates that porcine aminoacylase 1 is not active toward N-acyl-l-homoserine lactones as published previously but instead converts the autohydrolysis product N-acyl-l-homoserine.
  
  
  
  In this study, a novel method is presented for the identification, characterization, and optimization of quorum-quenching enzymes that are active toward N-acyl-l-homoserine lactones. These are the most common communication molecules among Gram-negative proteobacteria. The activity assay is a highly valuable complement to the available analytical tools in this field. It will facilitate studies on the environmental impact of quorum-quenching enzymes and contribute to the development of therapeutic and technical applications of this promising enzyme class.
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Greifswald, GermanyUniversity of California-Davis.
authors.forename: Daniel
authors.initials: D
authors.lastname: Last
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Greifswald, GermanyUniversity of California-Davis.
authors.forename: Georg H E
authors.initials: GH
authors.lastname: Krüger
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Greifswald, GermanyUniversity of California-Davis.
authors.forename: Mark
authors.initials: M
authors.lastname: Dörr
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Greifswald, GermanyUniversity of California-Davis uwe.bornscheuer@uni-greifswald.de.
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000581:Amidohydrolases / Q000032:analysis*; D001711:Biotransformation; D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D053038:Quorum Sensing
chemical_list: C088386:homoserine lactone; D002265:Carboxylic Ester Hydrolases; C432003:N-acyl homoserine lactonase; D000581:Amidohydrolases; C019665:amidase; D015107:4-Butyrolactone