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Open Access Research article

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies

Anke Steinbach1, Christine K Maurer1, Elisabeth Weidel3, Claudia Henn14, Christian Brengel1, Rolf W Hartmann12 and Matthias Negri1*

Author Affiliations

1 Helmholtz-Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123, Saarbrücken, Germany

2 Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123, Saarbrücken, Germany

3 ElexoPharm GmbH, Im Stadtwald A1.2, 66123, Saarbrücken, Germany

4 Current address: MIP Pharma GmbH, Kirkelerstr. 41, 66440,Blieskastel-Niederwürzbach, Germany

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BMC Biophysics 2013, 6:10  doi:10.1186/2046-1682-6-10

Published: 1 August 2013

Abstract

Background

PQS (Pseudomonas Quinolone 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.