Type III Polyketide Synthases: Discovery, Characterization, and Engineering

169 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.The polyketides are a diverse group of natural products with important applications in medicine and industry. Industry, especially the pharmaceutical industry, is under pressure to deliver "greener" chemical syntheses that are less environmentally damaging and incorporate renewable resources. There exists potential to replace current chemical syntheses of natural product or natural product-inspired pharmaceuticals with biological syntheses using enzymes. Polyketide biosynthesis is a particularly attractive target, as polyketide-derived products alone comprise 20% of the top-selling pharmaceuticals.This thesis describes our efforts to enable biosynthesis of polyketides via the discovery, characterization, and engineering of Type III PKS enzymes. Type III PKSs produce a wide array of aromatic structures in spite of their structural simplicity. Their products are often bioactive, and several compounds from plant Type III PKSs are under study for their health benefits. Herein we mined scientific literature and genomic data to discover these enzymes, and applied heterologous protein expression, protein crystallization, and protein and metabolic engineering strategies to understand and improve their properties.The enzyme PhlD was identified from scientific literature as a Type III PKS. We hypothesized that PhlD may be a phloroglucinol synthase, and observed phloroglucinol production when PhlD was expressed heterologously in E. coli. Protein and metabolic engineering were applied to improve phloroglucinol production, and achieved approximately four-fold increase in productivity to date. The enzyme ORAS from Neurospora crassa was initially identified as a putative Type III PKS in the sequenced genome of its host. We characterized the activity of ORAS and the crystal structure was solved and analyzed in collaboration with the laboratory of Professor Satish Nair at the University of Illinois, Urbana. Finally, we developed a screening system for the discovery of new Type III PKSs from medicinal plants. We created and validated a PCR-based screen for new Type III PKS enzymes from plants and also present preliminary data from screening the plants Eucalyptus camaldulensis and Eucalyptus robusta using this system.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C6-C7-C6 diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C6-C3 coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H2O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C6-C7-C6 scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes

A structure-based mechanism for benzalacetone synthase from Rheum palmatum

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 Å resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 Å resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel active-site pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase’s coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes