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J. Biol. Chem., Vol. 281, Issue 42, 32036-32047, October 20, 2006

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Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase from Pseudomonas fluorescens^*

From the Departments of Chemical and Biomolecular Engineering, and Chemistry, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

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 K_m = 13 ± 1 µM) 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 -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.

Received for publication, July 10, 2006 , and in revised form, August 23, 2006.

^* This work was supported by an Office of Naval Research Grant N00014-02-1-0725. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1.

¹ Supported by the United States National Science Foundation Graduate Research Fellowship Program.

² To whom correspondence should be addressed: Depts. of Chemical and Biomolecular Engineering and Chemistry, Center for Biophysics and Computational Biology, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-333-2631; Fax: 217-333-5052; E-mail: zhao5{at}uiuc.edu.

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