title: Ectopic expression and functional characterization of type III polyketide synthase mutants from Emblica officinalis Gaertn.
abstract: Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173 bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.
authors.affiliation: Plant Molecular Biology Division, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram, Kerala, 695014, India.
authors.forename: Girija
authors.initials: G
authors.lastname: Aiswarya
authors.affiliation: Plant Molecular Biology Division, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram, Kerala, 695014, India.
authors.forename: Vijayanathan
authors.initials: V
authors.lastname: Mallika
authors.affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, UK. lum@aber.ac.uk.
authors.forename: Luis A J
authors.initials: LA
authors.lastname: Mur
authors.affiliation: Plant Molecular Biology Division, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thiruvananthapuram, Kerala, 695014, India. evsoniya@rgcb.res.in.
authors.forename: Eppurathu Vasudevan
authors.initials: EV
authors.lastname: Soniya
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D018076:DNA, Complementary / Q000235:genetics; D000066630:Ectopic Gene Expression; D004591:Electrophoresis, Polyacrylamide Gel; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D055432:Metabolomics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010649:Phenylalanine / Q000235:genetics; D029842:Phyllanthus emblica / Q000201:enzymology* / Q000235:genetics*; D018515:Plant Leaves / Q000235:genetics; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D025341:Principal Component Analysis; D016415:Sequence Alignment; D014026:Nicotiana / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D010649:Phenylalanine; D048630:Polyketide Synthases
keywords: Chalcone synthase; Emblica officinalis; In vitro assay; Metabolomics; Site-directed mutagenesis