Substrate specificity, polyketide synthase

Reeves, C.D., Murli, S., Ashley, G.W. et al. (2001) Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry, 40, 15464. 

ABE, I., MORITA, H NOMURA, A., NOGUCHI, H., Substrate specificity of chalcone synthase enzymatic formation of unnatural polyketides from synthetic cinnamoyl-CoA analogues, J. Am. Chem. Soc., 2000,122,11242-11243. 

Recently, bacterial NRPS modules with the organization of A-KR-PCP have been discovered in the valino-mycin and cereulide synthetases. The A domains of these modules selectively activate a-keto acids. After the resulting adenylate is transferred to the PCP domain, the a-ketoacyl- -PCP intermediate is reduced to a PCP-bound, a-hydroxythioester by the KR domain. These domains use NAD(P)H as a cofactor and are inserted into A domains between two conserved core motifs analogous to MT domains. Their substrate specificity differs from that of polyketide synthase KR domains, which reduce /3-ketoacyl substrates. Similar fungal NRPSs, such as beauvericin synthetase, utilize A domains that selectively activate a-hydroxy acids. These molecules are thought to be obtained using an in trans KR domain, which directly reduces the necessary, soluble a-keto acid. 

R Pieper, G Luo, DE Cane, C Khosla. Remarkably broad substrate specificity of a modular polyketide synthase in a cell-free system. J Am Chem Soc 117 11373-11374, 1995. 

SF Haydock, JF Aparicio, I Molnar, T Schwecke, LE Khaw, A Konig, AFA Marsden, IS Galloway, J Staunton, PF Leadlay. Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA acyl carrier protein trans-acylase domains in modular polyketide synthases. FEBS Lett 374 246-248, 1995. 

To examine the function and substrate specificity of the polyketide-synthases, Khosla et al. devel-... 

Haydock SF, Aparicio JF, Mobiar I, Schwecke T, Khaw LE, Konig A, Marsden AF, Galloway IS, Staunton J, Leadlay PF (1995) Divergent Sequence Motifs Correlated with the Substrate Specificity of (Methyl) mMalonyl-CoA Acyl Carrier Protein Transacylase Domains in Modular Polyketide Synthases. FEBS Lett 374 246... 

Reeves CD, Murli S, Ashley GW, Piagentini M, Hutchinson CR, McDaniel R (2001) Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain Through Site-Specific Mutations. Biochemistry 40 15464... 

Tsai SC, Lu H, Cane DE, Khosla C, Stroud RM. Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. Biochemistry 2002 41 12598-12606. 

For in vitro studies of polyketide assembly using the purified components of the Type II PKS, it is essential to have the ACPs in their active holo-form which can then be acylated chemically or enzymatically (Scheme 30). The inactive apo-ACPs are converted to the active holo-form by addition of phosphopantet-heine from coenzyme A to a conserved serine residue. This addition is mediated in each organism by an ACP-holo synthase. In E, coli there is an ACP-holo synthase which is part of its endogenous fatty acid biosynthetic machinery but the substrate specificity of this enzyme for heterologous ACPs has been shown... 

Type III polyketide synthases (PKSs) generate a diverse array of natural products by condensing multiple acetyl units from malonyl-CoA to specific starter substrates (1,2). The homodimeric enzymes orchestrate a series of acyltransferase, decarboxylation, condensation, cyclization, and aromatizatidn reactions at two functionally independent active sites. Due to their ability to vary either the starter molecule or the type of cyclization and the number of condensations, they are, along with terpene synthases, one of the major generators of carbon skeleton diversity in plant secondary metabolites (i). Among the starter substrates used, benzoyl-CoA is a rare starter molecule. It is utilized by bacterial type I PKSs to form soraphen A, enterocin and the wailupemycins (4). in plants, benzoyl-CoA is the starter unit for two type III PKSs, benzophenone synthase (BPS) and biphenyl synthase (BIS), both of which were cloned in our laboratory. 

Figure 3. Polyketide synthase substrate specificity. Structures are shown for some of the substrates usedfor determining specificity of recombinant S. bicolor...

Predicting Modular Polyketide Synthase Acyl Transferase Domain Substrate Specificity... 

J. Lau, D. Cane, C. Khosla, Substrate specificity of the loading didomain of the erythromycin polyketide synthase, Biochemistry 2001, 29, 10514-10520. 

Stilbenes Polyketides (see) formed from one molecule of cinnamic acid and three molecules of malo-nyl-CoA. The immediate precursors of S. are the corresponding stilbenecarboxylic acids, which have a carboxyl group at C 2 of ring A (Fig.). Different S. synthases are specific for either cinnamoyl-CoA or p-coumaryl-CoA as substrates. Although S. synthase and chalcone synthase use the same substrates, they are distinct enzymes (antibodies to one do not cross-react with the other). S. synthase has been purified from cell suspension cultures of Arachis hypogaea... 

Funa N, Ohnishi Y, Ebizuka Y, Horinouchi S (2002) Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochem... 

Enzymatic syntheses within the microfluidic platform were also reported. The construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type HI polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microreactor were performed (Fig. 5) [11]. THNS immobilized to Ni-NTA agarose beads was prepacked into a microfluidic channel, while SBP was covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result was a tandem, two-step hiochip that enabled synthesis of novel polyketide derivatives. The first microchan-nel, consisting of THNS, resulted in the conversion of malonyl-CoA to flaviolin in yields of up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and... 

K. Watanabe, C.C.C. Wang, C.N. Boddy, D.E. Cane, C. Khosla, Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases. J. Biol. Chem. 278, 42020-42026 (2003)... 

G. Yadav, R.S. Gokhale, B. Mohanty, Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. J. Mol. Biol. 328, 335-363 (2003)... 

M. Jenner et al.. Substrate specificity in ketosynthase domains from Irani-AT polyketide synthases. Angew. Chem. Int. Ed. 52, 1143-1147 (2013)... 

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