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

Abstract

BackgroundPQS (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.ResultsEnzyme 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.ConclusionsEnzyme 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.

The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH‐like 2‐Heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) Biosynthesis Enzyme PqsBC

“…This view was later refined to the “open‐state model” by studies on mt FabH, which showed that a mutant with a blocked acyl‐binding pocket could still bind an inhibitor, but this was only possible if the enzyme opened by movement of the flap region and exposed its binding site . The open‐state model is further supported by molecular dynamics (MD) simulations of the FabH‐like PQS biosynthesis enzyme PqsD and ec FabH, which demonstrated large motions of the flap region and in the areas with conserved residues for adenine stacking . These MD profiles are strikingly similar to the structural fluctuations observed in the ensemble of PqsC crystal structures shown in Figure B.…”

PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal

“…Besides condensing anthraniloyl-CoA and malonyl-CoA or malonyl-ACP, PqsD can also mediate the condensation of anthraniloyl-CoA and 3-ketodecanoic acid to form HHQ ( 53 ). If PqsBC catalyzed the decarboxylative condensation of octanoyl-CoA and malonyl-CoA, 3-ketodecanoyl-CoA would be formed, so PqsBC might provide a substrate for PqsD-mediated HHQ synthesis.…”

Recent Advances in the Discovery of PqsD Inhibitors as Antimicrobial Agents

“…[17] PqsD is au nique prokaryotic structure due to the role of the QS pathway of P. aeruginosa in the expression of several virulence factors and biofilm formation. [17] PqsD is au nique prokaryotic structure due to the role of the QS pathway of P. aeruginosa in the expression of several virulence factors and biofilm formation.…”

“…Pseudomonas quinolone signal (PQS) and its precursor 4-hydroxy-2-heptylquinolone (HHQ) are signal molecules of the P. aeruginosa QS system, and the enzyme PqsD catalyzes the biosynthesis of HHQ. [17] PqsD is au nique prokaryotic structure due to the role of the QS pathway of P. aeruginosa in the expression of several virulence factors and biofilm formation. [18][19][20] PqsD belongs to the b-ketoacyl-ACP synthasef amily and is similari ns tructure to homologous b-ketoacyl-ACP synthase III (FabH) enzymes in Escherichia coli and Mycobacterium tuberculosis.…”