Steinbach Anke, Maurer Christine K, Weidel Elisabeth, Henn Claudia, Brengel Christian, Hartmann Rolf W, Negri Matthias
Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.
ElexoPharm GmbH, Im Stadtwald A1.2, 66123, Saarbrücken, Germany.
BMC Biophys. 2013 Aug 1;6(1):10. doi: 10.1186/2046-1682-6-10.
PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ.
Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and β-ketodecanoic acid.
Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for β-ketodecanoic acid were identified with distinct catalytic mechanism preferences.
PQS（铜绿假单胞菌喹诺酮信号）及其前体HHQ是铜绿假单胞菌群体感应系统的信号分子。它们通过与诱导毒力因子产生和生物膜形成的受体PqsR结合来发挥其在哺乳动物致病性中的作用。酶PqsD催化HHQ的生物合成。
采用酶动力学分析和表面等离子体共振（SPR）生物传感器实验来确定生物合成的机制和底物顺序。通过比较分析鉴定了参与PqsD功能的结构域。建立了动力学循环，并使用分子动力学（MD）模拟来研究PqsD动力学的分子基础。轨迹分析、口袋体积测量、结合能估计和分解确保了对底物邻氨基苯甲酰辅酶A和β-酮癸酸结合模式的深入了解。
酶动力学和SPR实验表明PqsD的乒乓机制是以ACoA作为第一个底物。对不同PqsD复合物的轨迹分析证明了配体依赖性诱导契合运动，影响修饰的ACoA漏斗进入二级通道的暴露。形成了一个隧道网络，其中Ser317通过与两种底物结合发挥重要作用。导致无活性S317F突变体的诱变实验证实了该残基的重要性。确定了β-酮癸酸的两种结合模式，具有不同的催化机制偏好。
