Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase fromPseudomonas fluorescens

Abstract

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 M) 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 C 4 -C 12 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.

Type III polyketide synthases in natural product biosynthesis

“…This tunnel assists the binding of long chain acyl-CoAs by means of dynamically increasing the volume of the active site cavity. The saturation mutagenesis on the tunnel successfully altered the substrate specificity of PhlD, which demonstrated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs and explained the high reactivity of PhlD with various substrates (30).…”

“…Finally, PKS18 from Mycobacterium tuberculosis has been shown to accept longchain aliphatic acid CoA-esters (C 6 -C 20 ) as the starter units to produce triketide or tetraketide pyrones via two or three condensation reactions (37). The crystal structure of PKS18 reveals a 20 Å substrate binding tunnel that allows this synthase to utilize a broad range of substrates, similar to PhlD (30). In addition to PKS18, several bacterial type III PKSs including PKS11 and SCO7671 from S. coelicolor were found to use long-chain fatty acid CoA-esters as the starter units (37).…”

Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

“…Homology modeling of PfPhlD using the structure of THNS as a reference highlighted the presence of a similar buried tunnel extending into the 'floor' of the active site cavity, which can potentially aid the binding of long chain acyl-CoAs [68]. The different reactivity towards phenylacetyl-CoA might suggest a larger and more flexible tunnel in PfPhlD that is capable of incorporating the bulky starter, compared to RppA.…”

“…These structural studies have provided insights into the mechanistic details of type III PKSs, illustrating how subtle modifications in the active site cavity can expand the biosynthetic repertoire and result in the generation of diverse products. Manipulation of active site residues by random mutagenesis [68] and/or site-directed mutagenesis has proved useful in the understanding of and subsequently, engineering of the substrate specificity and activity of type III PKSs.…”

Posttranscriptional Regulation of 2,4-Diacetylphloroglucinol Production by GidA and TrmE in Pseudomonas fluorescens 2P24

“…The locus contains four genes, phlA, phlC, phlB, and phlD, which are transcribed as a single operon (phlACBD) (13). The phlD gene encodes a type III polyketide synthase, which is responsible for the synthesis of phloroglucinol (PG) from three molecules of malonyl-coenzyme A (CoA) (17,18,19). The phlA, phlC, and phlB genes together are required for conversion of PG to monoacetylphloroglucinol (MAPG) and of MAPG to 2,4-DAPG (13,20).…”