Thioesterase polyketide

Gokhale, R.S., Hunziker, D., Cane, D.E. and Khosla, C. (1999) Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase. Chemistry Biology, 6, 117. 

Lu, H., Tsai, S.-C., Khosla, C. and Cane, D.E. (2002) Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase. Biochemistry, 41, 12590-12597. 

Boddy, C.N., Schneider, T.L., Hotta, K. et al. (2003) Epothilone C macrocyclization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase. Journal of the American Chemical Society, 125, 3428-3429. 

Phosphopantetheine tethering is a posttranslational modification that takes place on the active site serine of carrier proteins - acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs), also termed thiolation (T) domains - during the biosynthesis of fatty acids (FAs) (use ACPs) (Scheme 23), polyketides (PKs) (use ACPs) (Scheme 24), and nonribosomal peptides (NRPs) (use T domain) (Scheme 25). It is only after the covalent attachment of the 20-A Ppant arm, required for facile transfer of the various building block constituents of the molecules to be formed, that the carrier proteins can interact with the other components of the different multi-modular assembly lines (fatty acid synthases (FASs), polyketide synthases (PKSs), and nonribosomal peptide synthetases (NRPSs)) on which the compounds of interest are assembled. The structural organizations of FASs, PKSs, and NRPSs are analogous and can be divided into three broad classes the types I, II, and III systems. Even though the role of the carrier proteins is the same in all systems, their mode of action differs from one system to another. In the type I systems the carrier proteins usually only interact in cis with domains to which they are physically attached, with the exception of the PPTases and external type II thioesterase (TEII) domains that act in trans. In the type II systems the carrier proteins selectively interact... 

Figure 21-11 Catalytic domains within three polypeptide chains of the modular polyketide synthase that forms 6-deoxyerythronolide B, the aglycone of the widely used antibiotic erythromycin. The domains are labeled as for fatty acid synthases AT, acyltransferase ACP, acyl carrier protein KS, 3-ketoacyl-ACP synthase KR, ketoreductase DH, dehydrase ER, enoylreductase TE, thioesterase. After Pieper et al.338 Courtesy of Chaitan Khosla.

Figure 1 Polyketide biosynthesis. Polyketide backbones are formed via condensations from acyl-CoA thioesters of carboxylic acids. The (3-ketone which results from each condensation can undergo a series of reductive steps analogous to fatty acid biosynthesis. However, either none or only some of the reductive activities may occur in a given cycle. This allows PKSs to generate diversity through selection of priming and extender units, variation of the reductive cycle, and stereoselectivity. (ACP, acyl carrier protein AT, acyl transferase KS, ketosynthase DH, dehydratase ER, enoylreductase KR, ketoreductase TE, thioesterase.) The structure depicted in the lower right-hand corner is representative of the possible structural variations that can arise during polyketide biosynthesis.

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.

Figure 5 Domain organization of the erythromycin polyketide synthase. Putative domains are represented as circles and the structural residues are ignored. Each module incorporates the essential KS, AT, and ACP domains, while all but one include optional reductive activities. AT, acyltransferase ACP, acyl carrier protein KS, (3-ketoacyl synthase KR, P-ketoacyl reductase DH, dehydratase ER, enoyl reductase TE, thioesterase.

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.

FIGURE 19.4 Modular organization of the six modules (I—VI) of 6-deoxyerythronolide B synthase (DEBS) enzyme as derived from Saccharopolyspora erythraea. Enzyme activities are acyltransferases (AT), acyl carrier proteins (ACP), fi-ketoacyl-ACP synthases (KS), P-ketoreductases (KR), dehytratases (DH), enoyl reductases (ER), and thioesterases (TE). The TE-catalyzed release of the polyketide chain results in the formation of 6-dEB (70), 375 379 383... 

The molecular backbone of the antibiotic erythromycin A [6-desoxy-erythronolide B (3)] is built up repetitively from one propionyl-coenzyme A (1) and six methyl-malonyl-coenzyme A (2) constituents by the action of polyketide-synthase, which itself consists of three proteins (DEBS 1 -3) (Schemes 1 and 2). Each protein contains two modules with several separate, cat-alytically active domains. In the first section, DEBS 1 carries an additional loading zone, and DEBS 3 contains a thioesterase in the final segment, catalyzing the decoupling of the product by building the lactone ring [6],... 

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. 

J, Leadlay PF, Spencer JB. Evidence that a novel thioesterase is responsible for polyketide chain release during biosynthesis of the polyether ionophore monensin. ChemBioChem 2006 7 1435— 1442. 

Polyketide and non-ribosomal peptides produced by bacteria and fungi often attain the conformations that establish biological activity by cychzation constraints introduced by tailoring enzymes. This includes heterocychzation of cysteines, serines and threonines in non-ribosomal peptides. The second cychzation constraint is macrocychzation in polyketides, such as the above-mentioned antibiotic erythromycin and the antitumor epothilones. Regio- and stereospecific macrocychzation usuaUy occurs at the end of the polyketide and non-ribosomal peptide assembly hnes during chain release by thioesterase domains [49]. However, in the case of antibiotics of the ansamycin class, like the antitubercular drug rifamycin, the final... 

Polyketide formation in AF biosynthesis utilizes a hexanoyl CoA starter unit that is synthesized by aid of two specialized FASs (39) (Figure 4A). The genes for these FASs are transcribed from a common promoter region (40). The alpha subunit FAS has separate ACP and PP domains as well as KS and KR domains while the beta subunit has KS, AT, ACP and thioesterase (TE) domains (41). 

The AT homologue ZhuC in the R1128 PKS was found to be an acetyl-ACP thioesterase that is required to suppress acetate priming. In vitro PKS reconstitution experiments with ZhuC showed that its addition to the act and tcm minimal PKSs significantly attenuated the synthesis of acetate-primed polyketides (30). ZhuC was shown to catalyze the rapid hydrolysis of acetyl-ACP to yield acetate and holo-ACP (kct >150 min ). In contrast, ZhuC does... 

Figure 3. Priming and editing mechanisms for the RI128 initiation module. The acyl group carried by ZhuG is shuttled to the KS-CLF of the minimal PKS. The acyl-primed KS-CLF is then able to elongate the starter unit into a full length polyketide. ZhuC serves as an acetyl-ACP thioesterase.

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