Polyketides triketides

The avermectin PKS loading module has been used to generate a hybrid PKS system by replacing the loading module of DEBS 1-TE. The new hybrid PKS produced new hybrid polyketide (triketide lactones) which incorporated the isobutyrate and 2-methylbutyrate starter acids of avermectin biosynthesis, as well as the normal acetate and propionate starter units of erythromycin biosynthesis [51]. 

Kao, C.M., Luo, G.L., Katz, L. et al. (1995) Engineered biosynthesis of a triketide lactone from an incomplete modular polyketide synthase. Journal of the American Chemical Society, 117, 9105. 

Notably, natural variation in the type III PKS active site cavity, like that observed in Ipomoea and Petunia, does not result in functionally impaired enzymes, but in fact, generates catalytically active enzymes that display both altered substrate and product specificities. Sequential increases in the side chain volume of position 256 in alfalfa CHS2 result in decreases in polyketide chain length and predictable shifts in the ratio of tetraketide to triketide reaction products.32 These results functionally link the volume of the elongation/cyclization lobe in type III PKS to chain length determination. 

Abe T, Noma H, Noguchi H, Abe I (2006) Enzymatic formation of an unnatural methylated triketide by plant type III polyketide synthases. Tetrahedron Lett 47 8727-8730... 

Figure 9 Construction of bimodular polyketide synthases, (a) Chromosomal repositioning of the thioesterase domain from the C-terminus of module 6 to the end of module 2 in the erythromycin PKS leads to production of triketide lactones and the disruption of erythromycin biosynthesis, (b) DEBS 1-TE contains a fusion within the ACP domains of modules 2 and 6. In Saccharopolyspora erythraea and Streptomyces coelicolor the construct produced both propionate and acetate-derived lactones, (c) DEBS 1+TE contains a fusion between ACP2 and the thioesterase domain. In S. coelicolor, the protein biosynthesized the same lactones.

The overall for the synthesis of the Cg-lactone by DEBS 1+TE was measured to be 3.4 min [163]. For 6-dEB synthesis by complete DEBS, an apparent /Cjat of 0.5 min was determined. The measured of DEBS 1+TE indicates that this truncated PKS is highly active in a cell-free system and approaches catalytic activity comparable to in vivo levels. The apparent for (2S) methylmalonyl CoA consumption by DEBS 1+TE is 24 pM. Although starter units with shorter and longer side chains are incorporated into the respective triketide 6 lactones, DEBS 1+TE has a 7.5-fold preference for propionyl-CoA over butyryl-CoA and a 32-fold preference over acetyl-CoA. In the absence of the primer propionyl-CoA, DEBS 1+TE turns over (2S)-methylmalonyl CoA and NADPH at the same rate as in the presence of the starter unit. This suggests that DEBS 1+TE decar-boxylates the extender unit, transfers the evolving propionyl group to its active site KSl, and thus initiates polyketide chain elongation. 

Evidence for such a modular pathway has been provided from studies into the biosynthesis of the polyketide backbone 77 of (41 )-4-[(E)-2-butenyl]-4-methyl-L-threonine 78 which is incorporated into cyclosporin A in Tolypo-cladium niveum [117]. The proposed biosynthesis of 77 is presented in Scheme 29. In vitro studies using a cell extract have verified unambiguously that the biosynthetic mechanism is processive, that the first PKS free intermediate is the tetraketide 79, and that methylation unequivocally occurs at the stage of the enzyme bound 3-oxo-4-hexenoic acid thioester 80 which is the triketide product from the second elongation cycle. These and other results indicate that the methyl transferase activity is inherent in the second module of the putative PKS. 

Gane, D. E., Kudo, F., Kinoshita, K. Khosla, C. Precursor-directed biosynthesis biochemical basis of the remarkable selectivity of the erythromycin polyketide synthase toward unsaturated triketides. Chem. Biol. 9, 131—142 (2002). 

Die sequential coordinated action of the modular PKSs is illustrated in Eigiue 8.10, where (A) shows tlie 6-deoxyeiythronolide B synthase with catalytic domains, (B) depicts the production of triketide lactone, and (C) illustrates the rifaniycin synthetase, a polyketide synthase naturally primed by a nonribosomal PKS loading module which may be engineered to utiliize exogenous acids for the sythesis of substituted macrocycles. 

Polyketides. Collective name for natural products produced biosynthetically by way of poly(/5-oxo-carboxylic acids). The name was derived in 1907 by Collie on the basis of the hypothesis that natural prt ucts may be formed by multiplication of ketene (HjCsC o) units. The P. chains are constructed on multienzyme complexes (polyketide synthases) from acetyl- and malonyl-CoA ( acetogenins) or also by use of propio-nyl- and/or butyryl-CoA. Depending on the number of building blocks the natural products ate classified as triketides (n=3), tetraketides (n=4), etc. 

The a-methoxy-y-pyrone unit itself arises from the cychsation of three ketide units. The final three extension units condensed into the polyketide chain are left unmodified by reductive enzymes, giving a triketide which readily cyclises, with concurrent release from the acyl carrier protein. The immediate product is the y-hydroxy-a-pyrone 18. Tautomerisation to the y-pyrone tautomer 19 followed by an enzyme-mediated regioselective 0-methylation occurs to give the a-methoxy-y-pyrone 20 (Scheme 1.3) [12]. 

Alteration of Ghain Length. The last module of DEBS carries at the carboxyl terminus a thioesterase domain (TA), which releases the polyketide chain, forming the macrolactone. Repositioning the TA domain at the end of module 2, a triketide is formed, cyclized in a six-membered lactone (Cortes et al. 1995). By repositioning TA at the end of module 3, a tetraketide is formed that can cyclize in two ways, forming two different six-membered lactones (Kao et al. 1995). 

---