Substrate specificity, polyketide

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. 

Tang L, McDaniel R (2001) Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity. Chem Biol 8 547-555... 

Figure 6 (A) Reactions catalyzed by aromatic cyclases and aromatases. These enzymes control the diverse cyclization patterns of aromatic polyketides and in general display high regioselectivity and substrate specificity. (B) Examples of known products with different cyclization patterns that are accessible via the thioesterase (TE) domain of DEBS.

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. 

Three categories of synthetases are distinguished, based on their substrate specificity and mode of product synthesis. The two known types of polyketide synthetases (PKSs) (Type I and II) utilize acyl-coenzyme A (CoA) monomers while nonribosomal peptide synthetases (NRPSs) use amino acids and their analogs as substrates. Type I PKS and NRPS oligomerize these building blocks by a modular assembly-line arrangement while type II PKS iteratively assembles monomeric units. 

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... 

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

In further experiments, tcmO - a methyltransferase of the tetracenomycin cluster that methylates the C8-hydroxyl of tetracenomycin B3, an intermediate in the biosynthesis of tetracenomycin - was used to generate the new polyketide 3-O-Methyl-DMAC (59). Again, this experiment demonstrated that the variable substrate specificity of an enzyme increases its potential use in creating new molecules. 

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