Polyketide synthase enzymes

Polyketide synthase enzyme assays contained 100 mM potassium phosphate buffer (pH 7.0), 40 pM malonyl-CoA, 25 pM starter molecule (i.e. palmitoyl-CoA), and 2 pg protein in a 200 pL volume at 30° C for 30 minutes. Reactions were quenched by addition of 10 pL of 20% HCl. The lipid resorcinol products were extracted by phase partitioning with 1 ml of ethyl acetate. The organic phase (upper layer) obtained by centrifugation at -14,000 x g for 1 minute was transferred to a fresh tube, dried under vacuum, and subsequently analyzed by GC/mass spectrometry as a trimethysilyl derivative. Product formation was quantified using selected ion monitoring at m/z 268, a common fragment to all... 

Great advances have been made in the understanding of the role of multifunctional polyketide synthase enzymes (PKSs) in bacterial aromatic polyketide biosynthesis, and many such... 

Abell C, Bush BD, Staunton J (1986) Biomimetic Syntheses of the Polyketide Fungal Metabolites Altemariol and Rubrofusarin Models for Cyclisation Reactions Catalyzed by Polyketide Synthase Enzymes. J Chem Soc Perkin Trans 1 15... 

Meuter G, Hutchinson CR. Daunorubicin Type il polyketide synthase enzymes DpsA and DpsB determine neither the choice of starter unit nor the cyclization pattern of aromatic polyketides. ] Am Chem Soc 1995 117 5899-5900. 

It is very likely that a similar Type I polyketide synthase constructs the naphthoate fragment of azinomycin B. This will be a very interesting enzyme to study, since it will need to perform an unprecedented three regioselective reduction reactions, as well as controlling the polyketide chain length and directing its cycliza-tion. 

Until recently no enzymes able to produce olivetol-like compounds have been isolated. In an article by Puna et al., polyketide III enzymes were responsible for the formation of phenohc lipid compound [34], a natural product group that ohvetol belongs to. Although the biosynthesized compounds contained a longer chain, which increased over time, the study supported the hypothesis of olivetohc acid production by a polyketide III synthase. Further studies on the genetic and protein level are essential to elucidate the mode of mechanism by which olivetohc acid is formed in C. sativa. 

The biosynthesis of polyketides (including chain initiation, elongation, and termination processes) is catalyzed by large multi-enzyme complexes called polyketide synthases (PKSs). The polyketides are synthesized from starter units such as acetyl-CoA, propionyl-CoA, and other acyl-CoA units. Extender units such as malonyl-CoA and methylmalonyl-CoA are repetitively added via a decarboxylative process to a growing carbon chain. Ultimately, the polyketide chain is released from the PKS by cleavage of the thioester, usually accompanied by chain cyclization [49]. 

Pohl, N.L. (2002) Nonnatural substrates for polyketide synthases and their associated modifying enzymes. Current Opinion in Chemical Biology, 6, 773-778. 

In this chapter, we describe the atomic resolution structural elucidation of several plant type III polyketide synthases, including chalcone synthase, 2-pyrone synthase, and stilbene synthase. Manipulation of the catalytic activity and specificity of these biosynthetic enzymes by using a structurally guided approach offers a novel... 

LANZ T., TROPF, S., MARNER, F.J, SCHRODER, J., SCHRODER, G., The role of cysteines in polyketide synthases site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways, J. Biol. Chem., 1991, 266, 9971-9976. 

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

The Polyketide Pathway One metabolic process that has received much attention from natural product chemists is the production of secondary metabolites via polyketide synthase (PKS) enzymes. Polyketide synthases condense small starting... 

Native CHS is a homodimer with subunits of 40 to 44kDa. The structure of the protein produced from the CHS2 cDNA of M. sativa has been determined and the residues of the active site defined. It belongs to the polyketide synthase (PKS) group of enzymes that occur in bacteria, fungi, and plants, and is a type III PKS. All the reactions are carried out at a single active site without the need for cofactors. 

Preisig-Mueller, R. et al.. Plant polyketide synthases leading to stilbenoids have a domain catalyzing malonyl-CoA C02 exchange, malonyl-CoA decarboxylation, and covalent enzyme modification and a site for chain lengthening. Biochemistry, 36, 8349, 1997. 

Schroder, J., Plant polyketide synthases a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones. 

The biosynthesis of many hydroxylated natural products proceeds through regio- and enantioselective modification of polyketides, which are assembled through chain elongation via acetate or propionate units [2]. The enzymes responsible for the chain elongation and subsequent reduction, elimination, aromatiza-tion, and further modifications are classified as polyketide synthases [3]. These multifunctional enzymes have been used for whole-cell biotransformation toward unnatural metabolites that are within the scope of combinatorial biosynthesis... 

---