Characterization of the substrate specificity of PhlD, a type III polyketide synthase from Pseudomonas fluorescens

J Biol Chem. 2006 Oct 20;281(42):32036-47. doi: 10.1074/jbc.M606500200. Epub 2006 Aug 24.


PhlD, a type III polyketide synthase from Pseudomonas fluorescens, catalyzes the synthesis of phloroglucinol from three molecules of malonyl-CoA. Kinetic analysis by direct measurement of the appearance of the CoASH product (k(cat) = 24 +/- 4 min(-1) and Km = 13 +/- 1 microM) gave a k(cat) value more than an order of magnitude higher than that of any other known type III polyketide synthase. PhlD exhibits broad substrate specificity, accepting C4-C12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form C6-polyoxoalkylated alpha-pyrones from sequential condensation with malonyl-CoA. Interestingly, when primed with long chain acyl-CoAs, PhlD catalyzed extra polyketide elongation to form up to heptaketide products. A homology structural model of PhlD showed the presence of a buried tunnel extending out from the active site to assist the binding of long chain acyl-CoAs. To probe the structural basis for the unusual ability of PhlD to accept long chain acyl-CoAs, both site-directed mutagenesis and saturation mutagenesis were carried out on key residues lining the tunnel. Three mutations, M21I, H24V, and L59M, were found to significantly reduce the reactivity of PhlD with lauroyl-CoA while still retaining its physiological activity to synthesize phloroglucinol. Our homology modeling and mutational studies indicated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs. This suggested novel strategies for combinatorial biosynthesis of unnatural pharmaceutically important polyketides.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Catalysis
  • Cloning, Molecular
  • Kinetics
  • Models, Chemical
  • Models, Molecular
  • Molecular Structure
  • Mutation
  • Polyketide Synthases / chemistry
  • Protein Binding
  • Protein Conformation
  • Pseudomonas fluorescens / enzymology*
  • Substrate Specificity


  • Bacterial Proteins
  • PhlD protein, Pseudomonas
  • Polyketide Synthases