title: Mechanistic insights into the LsrK kinase required for autoinducer-2 quorum sensing activation.
abstract: In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV-vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s(-1)), Km,ATP (150 ± 30 μM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria.
authors.affiliation: The Skaggs Institute for Chemical Biology and Department of Chemistry and the Worm Institute for Research and Medicine (WIRM), the Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hixon
authors.forename: Daniel
authors.initials: D
authors.lastname: Globisch
authors.forename: Gunnar F
authors.initials: GF
authors.lastname: Kaufmann
authors.forename: Kim D
authors.initials: KD
authors.lastname: Janda
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006714:Homoserine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D007783:Lactones / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D053038:Quorum Sensing; D013056:Spectrophotometry, Ultraviolet
chemical_list: D029968:Escherichia coli Proteins; D007783:Lactones; C091088:N-octanoylhomoserine lactone; D009243:NAD; D006714:Homoserine; D000255:Adenosine Triphosphate; C549217:LsrK protein, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor)