title: Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Steinbach
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.
authors.forename: Christine K
authors.initials: CK
authors.lastname: Maurer
authors.affiliation: ElexoPharm GmbH, Im Stadtwald A1.2, 66123, Saarbrücken, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Weidel
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.|Current address: MIP Pharma GmbH, Kirkelerstr. 41, 66440,Blieskastel-Niederwürzbach, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Henn
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Brengel
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.|Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123, Saarbrücken, Germany.
authors.forename: Rolf W
authors.initials: RW
authors.lastname: Hartmann
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Negri
references.citation: Nat Prod Rep. 2002 Oct;19(5):581-96
references.pmid: 12430724
references.citation: J Bacteriol. 2008 Aug;190(15):5339-52
references.pmid: 18539738
references.citation: FEMS Microbiol Rev. 2005 Nov;29(5):935-47
references.pmid: 16219513
references.citation: Int J Med Microbiol. 2006 Apr;296(2-3):73-81
references.pmid: 16476569
references.citation: Biochemistry. 2009 Sep 15;48(36):8644-55
references.pmid: 19694421
references.citation: FEMS Microbiol Lett. 2002 Sep 24;215(1):41-6
references.pmid: 12393198
references.citation: Mol Biosyst. 2008 Sep;4(9):882-8
references.pmid: 18704225
references.citation: Anal Biochem. 2012 Sep 1;428(1):28-30
references.pmid: 22677626
references.citation: Trends Biochem Sci. 2006 Jan;31(1):64-71
references.pmid: 16356722
references.citation: Proteins. 2006 Nov 15;65(3):712-25
references.pmid: 16981200
references.citation: Chem Biol. 2012 Mar 23;19(3):381-90
references.pmid: 22444593
references.citation: Acc Chem Res. 2000 Dec;33(12):889-97
references.pmid: 11123888
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: Microbiology (Reading). 2008 Aug;154(Pt 8):2184-2194
references.pmid: 18667552
references.citation: Anal Bioanal Chem. 2007 Jan;387(2):409-14
references.pmid: 17019573
references.citation: J Bacteriol. 2005 Jun;187(11):3630-5
references.pmid: 15901684
references.citation: Mol Microbiol. 2005 Feb;55(4):998-1014
references.pmid: 15686549
references.citation: J Bacteriol. 2002 Dec;184(23):6472-80
references.pmid: 12426334
references.citation: J Biol Chem. 2001 Mar 16;276(11):8231-8
references.pmid: 11078736
references.citation: FEBS Lett. 2009 Sep 3;583(17):2939-46
references.pmid: 19665020
references.citation: J Mol Biol. 1997 Apr 4;267(3):727-48
references.pmid: 9126849
references.citation: Structure. 2000 Feb 15;8(2):185-95
references.pmid: 10673437
references.citation: Int J Med Microbiol. 2006 Apr;296(2-3):83-91
references.pmid: 16483840
references.citation: Curr Opin Microbiol. 2009 Apr;12(2):182-91
references.pmid: 19249239
references.citation: J Bacteriol. 1991 May;173(9):3000-9
references.pmid: 1902216
references.citation: Chem Biol. 2008 Apr;15(4):402-12
references.pmid: 18420147
references.citation: Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6424-8
references.pmid: 7604006
references.citation: FEMS Microbiol Ecol. 2011 Aug;77(2):413-28
references.pmid: 21539583
references.citation: Nucleic Acids Res. 2000 Jan 1;28(1):235-42
references.pmid: 10592235
references.citation: J Biol Chem. 2000 Sep 8;275(36):28201-7
references.pmid: 10840036
references.citation: J Comput Aided Mol Des. 2011 Apr;25(4):371-93
references.pmid: 21516317
references.citation: Protein Sci. 2005 Aug;14(8):2087-94
references.pmid: 15987898
references.citation: PLoS Pathog. 2007 Sep 14;3(9):1229-39
references.pmid: 17941706
references.citation: Microbiol Mol Biol Rev. 2012 Mar;76(1):46-65
references.pmid: 22390972
references.citation: Genome Res. 2005 Feb;15(2):330-40
references.pmid: 15687296
references.citation: Bioinformatics. 2011 Dec 1;27(23):3276-85
references.pmid: 21967761
references.citation: Proteins. 2008 Nov 1;73(2):299-319
references.pmid: 18433058
references.citation: Proteins. 2003 Sep 1;52(4):609-23
references.pmid: 12910460
references.citation: J Comput Aided Mol Des. 2011 Sep;25(9):795-811
references.pmid: 21822722
references.citation: J Am Chem Soc. 2012 Oct 3;134(39):16143-6
references.pmid: 22992202
references.citation: PLoS One. 2010 Apr 12;5(4):e10115
references.pmid: 20404933
references.citation: PLoS One. 2010 Aug 10;5(8):e12026
references.pmid: 20706575
references.citation: J Biol Chem. 2008 Oct 24;283(43):28788-94
references.pmid: 18728009
references.citation: J Mol Biol. 2005 Mar 11;346(5):1313-21
references.pmid: 15713483
references.citation: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14613-8
references.pmid: 11724939
references.citation: Bioinformatics. 2009 May 1;25(9):1189-91
references.pmid: 19151095
references.citation: Chembiochem. 2011 Apr 11;12(6):850-3
references.pmid: 21425231
references.citation: Mol Microbiol. 2006 Dec;62(6):1689-99
references.pmid: 17083468
references.citation: Proteins. 2008 Mar;70(4):1219-27
references.pmid: 17847101