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.

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis

“…Type III PKSs are promiscuous enzymes that have a broad tolerance for diverse substrates and are able to catalyze multiple reactions [84,85]. Type III PKSs that use cyclic nitrogen-containing substrates have been previously characterized for their roles in alkaloid production [86][87][88]. However, unlike these previous studies the predicted substrate in TA metabolism, N-methyl-∆ 1 -pyrrolinium cation (19) [81].…”

Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

“…The 'gatekeeper' Phe215 is generally conserved in most type III PKSs and is located between the CoA-binding tunnel and the entrance of the active site cavity. Despite having a lower malonyl-CoA decarboxylation activity compared to wild-type alfalfa CHS, the F215S mutant is able to utilize p-coumaroyl-CoA, phenylacetyl-CoA, benzoyl-CoA, and C5-C8 acyl-CoAs with less than 3% the catalytic efficiency of the wild-type enzyme to produce tetraketide lactones [100]. Intriguingly, although N-methylanthraniloyl-CoA is not normally utilized by the wild type CHS, the F215S mutant was found to accept the bulky starter with a comparable k cat /K m to R. graveolens acridone synthase (ACS), to form a tetraketide N-methylanthraniloyltriacetic acid lactone (31).…”

“…Simultaneous mutation of the neighbouring Phe66 residue to leucine resulted in an OKS double mutant that is capable of synthesizing an unnatural dodecaketide naphthophenone TW95a (70) by the successive decarboxylative condensations of 12 units of malonyl-CoA, making it one of the longest polyketide synthesized by the structurally simple type III PKS (Scheme 24C) [104]. having a lower malonyl-CoA decarboxylation activity compared to wild-type alfalfa CHS, the F215S mutant is able to utilize p-coumaroyl-CoA, phenylacetyl-CoA, benzoyl-CoA, and C5-C8 acyl-CoAs with less than 3% the catalytic efficiency of the wild-type enzyme to produce tetraketide lactones [100]. Intriguingly, although N-methylanthraniloyl-CoA is not normally utilized by the wild type CHS, the F215S mutant was found to accept the bulky starter with a comparable kcat/Km to R. graveolens acridone synthase (ACS), to form a tetraketide N-methylanthraniloyltriacetic acid lactone (31).…”

Molecular analysis of a type III polyketide synthase gene in Fallopia multiflora

“…CHS and STS are the most investigated members at both the biochemical and the molecular levels (Flores-Sanchez & Verpoorte 2009). Despite the functional diversity, plantspecific PKS proteins have in common to be dimeric proteins of approximately 42 kDa with a catalytic triad Cys-His-Asn in the active centre (Jez et al 2002). This facilitates the isolation of new genes coding for PKS family proteins based on homology-based techniques, such as using degenerate oligonucleotide primers.…”