Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity

Abstract

Type III polyketide synthases (PKS) generate an array of natural products by condensing multiple acetyl units derived from malonyl-CoA to thioester-linked starter molecules covalently bound in the PKS active site. One strategy adopted by Nature for increasing the functional diversity of these biosynthetic enzymes involves modifying polyketide assembly by altering the preference for starter molecules. Chalcone synthase (CHS) is a ubiquitous plant PKS and the first type III PKS described functionally and structurally. Guided by the three-dimensional structure of CHS, Phe-215 and Phe-265, which are situated at the active site entrance, were targeted for site-directed mutagenesis to diversify CHS activity. The resulting mutants were screened against a panel of aliphatic and aromatic CoA-linked starter molecules to evaluate the degree of starter molecule specificity in CHS. Although wild-type CHS accepts a number of natural CoA thioesters, it does not use N-methylanthraniloyl-CoA as a substrate. Substitution of Phe-215 by serine yields a CHS mutant that preferentially accepts this CoA-thioester substrate to generate a novel alkaloid, namely N-methylanthraniloyltriacetic acid lactone. These results demonstrate that a point mutation in CHS dramatically shifts the molecular selectivity of this enzyme. This structure-based approach to metabolic redesign represents an initial step toward tailoring the biosynthetic activity of plant type III PKS.

Figure 1

Overview of plant type III polyketide synthases. (A) Reactions catalyzed by CHS and ACS. Only the starter molecules are shown. Both enzymes use three molecules of malonyl-CoA during the elongation of the starter molecule. Chalcone is on top and acridone is on the bottom. (B) Ribbon diagram of the CHS homodimer showing the catalytic residues (Cys-164, His-303, and Asn-336) and the two phenylalanines (Phe-215 and Phe-265) at the boundary between the CoA (gold) binding site and the active site of one monomer. Figure prepared with molscript (35) and pov-ray [POV-Team (1997) pov-ray, Persistence of Vision Ray-Tracer; http://www.povray.org].

Figure 2

Thin-layer chromatography screen for activity with different starters. By using standard assay conditions, 10 μg of either wild-type CHS (A), F265V mutant (B), F215S mutant (C), or ACS (D) was incubated for 30 min with various starters and [2-¹⁴C]malonyl-CoA. The starter molecule for each reaction is indicated above the lanes. The position of naringenin in lane 1 is indicated for CHS and the F265V mutant. The positions of p-coumaroyltriacetic acid lactone in lane 1 and the new product in lane 9 are indicated for the F215S mutant. The position of acridone in lane 9 is indicated for ACS.

Figure 3

LC/MS/MS analysis of the major reaction products of ACS and CHS F215S. (A) Fragmentation pattern and chemical structure of the major product acridone obtained by using N-methylanthraniloyl-CoA and malonyl-CoA as substrates in a large scale reaction with ACS. (B). The analogous LC/MS/MS analysis as carried out in A but now using the CHS F215S mutant.

Figure 4

Structure of the F215S mutant active site and model of starter molecule binding. (A) Stereo-view illustrates the active site of the F215S mutant, including the catalytic residues (Cys-164, His-303, and Asn-336) and Phe-265. Both conformers of Ser-215 are shown. The CoA-thioester extends from the left side of the view. N-methylanthraniloyl-CoA has been modeled by applying the structural restraints discussed in the text. (B) Stereo-view of the wild-type CHS active site with N-methylanthraniloyl-CoA modeled at the entrance. Steric clashes with Phe-215 prevent the CoA-thioester from adopting the conformation depicted in A. (C) Stereo-view of the wild-type CHS active site with p-coumaroyl-CoA modeled at the entrance highlighting the ability of the propanoid moiety to extend the phenolic ring deep into the active site.