Synthase rapamycin

B. Erythromycin, Rapamycin, and Other Modular Polyketide Synthases... 

Macrolides and polyethers such as erythromycin A (4), FK 506, rapamycin or avermectin A (5, Scheme 1) are products of modular type I polyketide-synthases. These compounds are distinguished by extraordinary structural diversity and complexity [1,2]. Because of their biological potency, members of this structural class as well as the aromatic polycyclic products of type II polyketide-synthases, tetracyclines and anthara-cyclines, e.g. adriamycin (6), became useful as pharmaceuticals (antibiotics, cytostatics, immunosuppressives) [1,2],... 

Attur MG, Patel R,ThakkerG,Vyas P, Levartovsky D, Patel P, Naqvi S, Raza R, Patel K, Abramson D, Bruno G, Abramson SB, Amin AR. Differential antl-Inflammatory effects of immunosuppressive drugs cyclosporin, rapamycin and FK-506on inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and PGE2 production. Inflamm Res 2000 49 20-26. 

Lin AL, Zheng W, Halloran JJ, Burbank RR, Hussong SA, Hart MJ et al (2013) Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer s disease. J Cereb Blood Flow Metab 33 1412-1421... 

Many important therapeutics, in use in clinics today, are biosynthesized by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) paradigm. For example, many of the antibiotics (penicillin, cephalosporin, vancomycin, erythromycin, etc.), immunosuppressors (cyclosporine, rapamycin), antiviral agents (luzopeptin A), antitumor agents (bleomycin), and toxins (thaxtomin) are NRPS and PKS derived.20-22 Figure 1 displays a small selection of natural products that are NRPS and PKS derived and illustrates the diversity of molecular structures generated by these biosynthetic paradigms. 

Keywords Aliphatic polyketides Biosynthesis Polyketide Synthase Genetic Engineering Erythromycin A Rapamycin. 

D-alanine residue. Other polyketides which contain such an arrangement are the trienomycins [99], while similar CHC chain terminating (synthase starter) units are found in a branch of asukamycin [96], and in the eo-cyclohexyl fatty acids of certain thermophilic bacteria [100] substituted CHCs are also found as PKS starter units in the rapamycin family of polyketides (see Sect. 8). The cyclohexyl moieties of these compounds have been demonstrated to derive from the shiki-mate pathway. 

The polyketides are a family of natural products containing many important pharmaceutical agents that are synthesized through the multienzyme complex, polyketide synthase, which can display substantial molecular diversity with respect to chain length, monomer incorporated, reduction of keto groups, and stereochemistry at chiral centers (189). This variability, together with the existence of several discrete forms of polyketide synthase, allows the generation of diverse structures like erythromycin, avermectin, and rapamycin. This biochemical diversity has been considerably expanded by the introduction of new sub strate species that were used by the enzymes to produce new or unnatural polyketides (190). 

Fig. 15. Predicted domain organization and biosynthetic intermediates of the rapamycin synthase. Each circle represents an enzymatic domain as defined in Figs. 5 and 12. PS, nonribosomal peptide synthetase. (Continued)...

Aparicio, J. E, Molar, I., Schwecke, T., Kdnig, A., Haydock, S. F., Khaw, L. E., Staunton, J., and Leadlay, P. F. (1996). Organization of the biosynthetic gene cluster for rapamycin in Strep-tomyces hygwscopicus Analysis of the enzymatic domains in the modular polyketide synthase. Gene 169,9-16. 

Manipulation of the DEBS system has led to the most impressive demonstration of combinatorial biosynthesis to date. McDaniel and coworkers have utilized specific module-swapping strategies to access a variety of 6-deoxyerythronolide B analogs with modifications at each carbon of the macrolide backbone [26]. Modules 1-6 of DEBS were systematically replaced with individual rapamycin synthase components to alter oxidation state and methylation in the final polyketide product. The study produced 60 unique structures at yields ranging from 1 to 70% of that of 6-deoxyerythronolide B (Fig. 9.2-5). However, each new compound required independent synthase engineering, which made library construction quite tedious. 

Strain produced 6-deoxyerythronolide B. This polyketide synthase was then used for a variety of experiments in which modules/domains of the polyketide synthase were exchanged. As an example, compounds produced hy the substitution of KR domains are shown in Fig. 5. In these examples, KR domains from the erythromycin PKS have been replaced by domains from the rapamycin producer [71]. 

An enormous range of medically important polyketide and peptide natural products assembled by modular polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and mixed PKS/NRPS systems have macrocyclic structures, including the antibiotics erythromycin (PKS) and daptomycin (NRPS), the immunosuppressants cyclosporin (NRPS) and rapamycin (PKS/NRPS), and the antitumor agent epothilone (PKS/NRPS). PKSs and NRPSs are large, multifunctional proteins that are organized into sets of fnnc-tional domains termed modules. The order of modules corresponds directly to the seqnence of monomers in the product. Synthetic intermediates are covalently tethered by thioester linkages to a carrier protein domain in each module. The thiol tether on each carrier domain is phosphopantetheine, which is attached to a conserved serine residne in the carrier protein in a post-translational priming reaction catalyzed by a phosphopantetheinyltransferase. 

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