Ass of PqsD inhibitors that reduce biofilm and virulence factor PubMed…

aeruginosa validates PqsD as a target for the development of anti-infectives [22]. PqsD is a homodimeric bi-substrate enzyme with high structural similarity to FabH and other -ketoacyl-[ACP] synthases III (KAS III). They share a common thiolase fold (), a long tunnel to the active site, and the same catalytic residues [23-25]. Three PDB structures of PqsD exist [26]: as apoform (3H76), as Cys112-ligated anthranilate (CSJ) complex with ACoA molecules in the primary funnel (3H77) and as Cys112Ala mutant in complex with anthranilic acid (3H78) [23]. In all three structures the catalytic centre is accessible by two channels in L-shape: the primary CoA/ACP-funnel, and the shorter secondary channel (Additional file 1: Figure. SI1). However, the molecular details of ACoA access and, in Methyl 3-amino-2-chlorobenzoate particular, the binding mode and the subsequent incorporation of K are unknown. Knowledge of the kinetics and of the conformational flexibility of an enzyme can significantly contribute to a successful rational drug design [27-29]. Herein we study the molecular basis of PqsD and the HHQ biosynthesis combining experimental and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10572343 in silico methods. Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine the mechanism and the substrate order of the biosynthesis; comparative analysis of PqsD to homologous KAS-III enzymes was useful to identify domains specific for PqsD functionality. Molecular dynamics (MD) simulations were carried out to explore the binding modes of ACoA and K as well as the conformational flexibility of PqsD.obtained (data not shown). The initial velocity (v) was calculated by dividing the product concentration by the reaction time. Plotting the data with GraphPad Prism 5 software resulted in an array of parallel lines in the Lineweaver-Burk-Plot and a common Y-axis-intercept in the Hanes-Woolf-Plot (Figure 1A and 1B). This suggests a ping pong kinetic mechanism for PqsD as described also for other Claisen condensing enzymes [30]. The results were in agreement with reported mass spectrometric [31], structural [23], and surface plasmon resonance (SPR) based [32] studies revealing the formation of an anthraniloyl-PqsD intermediate with concomitant release of the first product CoA before binding of the second substrate -ketodecanoic acid (K). The KM data (ACoA 0.875 ?.140 M, K 1300 ?158 M) correlate well with the KD values determined with SPR by our group (ACoA 1.08 M, K 2.95 mM) [32]. Also, the kinetic parameters, derived mutually varying both substrates (Figure 1C) and fitting the data with the ping-pong Equation (1) are within the range of the apparent values determined by Pistorius et al. (KM app, = Methyl 3-amino-2-chlorobenzoate 598.5 ?106 M; Vmax = 495.8 ?37.5 fmol HHQ/min/pmol PqsD; kcat (PqsD as monomer) = 0.01 s -1). CoA (1) [33] SPR biov ?VmaxKm ACoA��ACoA 1?Km KResults and discussion Knowledge of enzyme kinetics for multi-substrate reactions is helpful to set up and interpret MD simulations. We performed biochemical and biophysical studies to determine the underlying kinetic mechanism of PqsD.Biochemical and biophysical characterization hint at ping-pong kinetic mechanism of PqsDEnzyme kinetic studies were performed using a 96-well format-based in vitro assay with the purified enzyme PqsD to determine the kinetic parameters for each substrate. Optimum enzymatic reaction conditions were determined in advance. Plotting product formation versus.