Empting M

References (3)

Title : Mechanistic details for anthraniloyl transfer in PqsD: the initial step in HHQ biosynthesis - Hutter_2014_J.Mol.Model_20_2255
Author(s) : Hutter MC , Brengel C , Negri M , Henn C , Zimmer C , Hartmann RW , Empting M , Steinbach A
Ref : J Mol Model , 20 :2255 , 2014
Abstract : PqsD mediates the conversion of anthraniloyl-coenzyme A (ACoA) to 2-heptyl-4-hydroxyquinoline (HHQ), a precursor of the Pseudomonas quinolone signal (PQS) molecule. Due to the role of the quinolone signaling pathway of Pseudomonas aeruginosa in the expression of several virulence factors and biofilm formation, PqsD is a potential target for controlling this nosocomial pathogen, which exhibits a low susceptibility to standard antibiotics. PqsD belongs to the beta-ketoacyl-ACP synthase family and is similar in structure to homologous FabH enzymes in E. coli and Mycobacterium tuberculosis. Here, we used molecular dynamics simulations to obtain the structural position of the substrate ACoA in the binding pocket of PqsD, and semiempirical molecular orbital calculations to study the reaction mechanism for the catalytic cleavage of ACoA. Our findings suggest a nucleophilic attack of the deprotonated sulfur of Cys112 at the carbonyl carbon of ACoA and a switch in the protonation pattern of His257 whereby Ndelta is protonated and the proton of Nsigma is shifted to the sulfur of CoA during the reaction. This is in agreement with the experimentally determined decreased catalytic activity of the Cys112Ser mutant, whereas the Cys112Ala, His257Phe, and Asn287Ala mutants are all inactive. ESI mass-spectrometric measurements of the Asn287Ala mutant show that anthraniloyl remains covalently bound to Cys112, thus further supporting the inference from our computed mechanism that Asn287 does not take part in the cleavage of ACoA. Since this mutant is inactive, we suggest instead that Asn287 must play an essential role in the subsequent formation of HHQ in vitro.
ESTHER : Hutter_2014_J.Mol.Model_20_2255
PubMedSearch : Hutter_2014_J.Mol.Model_20_2255
PubMedID: 24842325

Title : From in vitro to in cellulo: structure-activity relationship of (2-nitrophenyl)methanol derivatives as inhibitors of PqsD in Pseudomonas aeruginosa - Storz_2014_Org.Biomol.Chem_12_6094
Author(s) : Storz MP , Allegretta G , Kirsch B , Empting M , Hartmann RW
Ref : Org Biomol Chem , 12 :6094 , 2014
Abstract : Recent studies have shown that compounds based on a (2-nitrophenyl)methanol scaffold are promising inhibitors of PqsD, a key enzyme of signal molecule biosynthesis in the cell-to-cell communication of Pseudomonas aeruginosa. The most promising molecule displayed anti-biofilm activity and a tight-binding mode of action. Herein, we report on the convenient synthesis and biochemical evaluation of a comprehensive series of (2-nitrophenyl)methanol derivatives. The in vitro potency of these inhibitors against recombinant PqsD as well as the effect of selected compounds on the production of the signal molecules HHQ and PQS in P. aeruginosa were examined. The gathered data allowed the establishment of a structure-activity relationship, which was used to design fluorescent inhibitors, and finally, led to the discovery of (2-nitrophenyl)methanol derivatives with improved in cellulo efficacy providing new perspectives towards the application of PqsD inhibitors as anti-infectives.
ESTHER : Storz_2014_Org.Biomol.Chem_12_6094
PubMedSearch : Storz_2014_Org.Biomol.Chem_12_6094
PubMedID: 24909330

Title : Biochemical and biophysical analysis of a chiral PqsD inhibitor revealing tight-binding behavior and enantiomers with contrary thermodynamic signatures - Storz_2013_ACS.Chem.Biol_8_2794
Author(s) : Storz MP , Brengel C , Weidel E , Hoffmann M , Hollemeyer K , Steinbach A , Muller R , Empting M , Hartmann RW
Ref : ACS Chemical Biology , 8 :2794 , 2013
Abstract : Antivirulence strategies addressing bacterial pathogenicity without exhibiting growth inhibition effects represent a novel approach to overcome today's crisis in antibiotic development. In recent studies, we examined various inhibitors of PqsD, an enzyme involved in formation of Pseudomonas aeruginosa cell-to-cell signaling molecules, and observed desired cellular effects for 2-nitrophenyl derivatives. Herein, we investigated the binding characteristics of this interesting compound class using several biochemical and biophysical methods. The inhibitors showed time-dependent activity, tight-binding behavior, and interactions with the catalytic center. Furthermore, isothermal titration calorimetry (ITC) experiments with separated enantiomers revealed contrary thermodynamic signatures showing either enthalpy- or entropy-driven affinity. A combination of site-directed mutagenesis and thermodynamic profiling was used to identify key residues involved in inhibitor binding. This information allowed the proposal of experimentally confirmed docking poses. Although originally designed as transition state analogs, our results suggest an altered position for both enantiomers. Interestingly, the main difference between stereoisomers was found in the orientation of the hydroxyl group at the stereogenic center. The predicted binding modes are in accordance with experimental data and, thus, allow future structure-guided optimization.
ESTHER : Storz_2013_ACS.Chem.Biol_8_2794
PubMedSearch : Storz_2013_ACS.Chem.Biol_8_2794
PubMedID: 24099650