Biosynthesis cyclosporin

It should be noted that while TE domains represent the most common solution in releasing macrocyclic NRPs and PKs, other pathways are known. For instance, in the biosynthesis of cyclosporine, the cyclization is proposed to be catalyzed by the most downstream C-domain [48]. Macrocyclization can also occur under reduction of a carbonyl group mediated by a reduction domain (R-domain) as proposed in the synthesis of the macrocyclic imine nostocyclopeptide [49]. The synthetic utility of these cyclization strategies has not yet been reported. 

The immunosuppressant compound170 FK-506, similar in effect to cyclosporin A, the leading drug for use in immune system suppression to prevent rejection of transplanted organs171, has been labelled at carbon atoms 10, 16, 18, 21a, 24 and 26 by fermentative biosynthesis using sodium [l-14C]propionate as a precursor172. The same 13C-labelled positions were derived from [l-13C]propionate. FK-506 producing culture Streptomyces tsukubaenis no 9993 has been utilized in this biosynthesis (120 h incubation at 29 °C). 

In a limited number of NRPSs, the final module terminates in a specialized C domain that catalyzes chain release through amide bond formation. Modules of this type are found in the synthetases involved in the biosynthesis of enniatin, vibriobactin, cyclosporin/ HC-toxin/ and PF1032A. Unlike TE termination, this method of chain release does not utilize an acyl-ester intermediate. Most likely, the chain termination precursor is presented to the C domain as an aminoacyl-5-PCP substrate. Most of these specialized C domains... 

Kleinkauf, H. von Dohren, H. (1997) Biosynthesis of cyclosporins and related peptides. In Fungal Biotechnology. Anke, T., ed.. Chapman Hall, London, pp. 147-61. 

Parallel with chemical investigations, microbiological studies were intensified, especially the search for high producing mutants, as well as the improvement of media and culture conditions to enhance yields. A substantial feedback resulted from examination of the biosynthesis of cyclosporins. Yields and the composition of the metabolite mixture were significantly influenced by exogenously supplied amino-acid precursors. 

A non-ribosomal biosynthetic pathway is clearly indicated for cyclosporin A, considering the uncommon structural elements MeBmt, L-a-aminobutyric acid and D-alanine as well as the plethora of isolated congeners [20,21]. Non-ribosomal biosynthesis directed by multienzyme thiotemplates have been reported for other small peptides of microbial origin, for example, gramicidin S [22] and enniatin [23]. Experimental data for cyclosporin A were obtained by feeding appropriate labelled precursors to cultures of T. inflation strains. The distribution profile of the labelled atoms in cyclosporin A was determined by 3H- or 13C-NMR spectroscopy. In preliminary trials with several tritium and carbon-14 labelled precursors, [met/y>/-3H]methionine proved to be the most suitable marker for the biosynthetic preparation of radiolabelled cyclosporin A for pharmacokinetic and metabolic studies [24],... 

In more recent investigations, the assumed multienzyme involved in cyclosporin A biosynthesis could be isolated from T. inflatum. A partially purified enzyme fraction was indeed capable of forming enzyme-substrate complexes by thioester linkage. Although de novo synthesis (in vitro) of cyclosporin A has not yet been achieved, the formation of a partial sequence, namely, the diketopiperazine cyclo(DAla-MeLeu), from D-alanine and L-leucine was observed under consumption of ATP and S-adenosyl-L-methio-nine [25]. 

The incorporation of constitutional and foreign amino acids under replacement of inborn building units demonstrates convincingly the low specificity in the biosynthesis of cyclosporins characteristic for a non-ribosomal biosynthetic pathway. 

Table 1.4. IMMUNOSUPPRESSIVE ACTIVITY OF CYCLOSPORINS nat = natural dbs = by directed biosynthesis ps = by partial synthesis syn = by total synthesis + + +, strong immunosuppressive activity + +, moderate activity +, weak activity (+) or -, no significant activity.

Lawen A (1996) Biosynthesis and mechanism of action of cyclosporins. Prog Med Chem 33, 53-97. 

M Offenzeller, G Santer, K Totschnig, Z Su, H Moser, R Traber, E Schneider-Scherzer. Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway. Biochemistry 35 8401-8412, 1996. 

Studies made by feeding experiments with 14C-labeled precursors showed that the biosynthesis of cyclosporins proceeds by a thioltemplate mechanism, which, owing to the A-mcthylating steps, has strong resemblance to that of enniatin... 

K Hoffmann, E Schneider-Scherzer, H Kleinkauf, R Zocher. Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis. J Biol Chem 17 12710-12714, 1994. 

R Zocher, N Madry, H Peeters, H Kleinkauf. Biosynthesis of cyclosporin A. Phytochemistry 23 549-551, 1984. 

J Dittmann, RM Wenger, H Kleinkauf, A Lawen. Mechanism of cyclosporin A biosynthesis. J Biol Chem 269 2841-2846, 1994. 

M Glinski. Biosynthesis of cyclosporin A and enniatin B. Ph.D. thesis, Technische Universitat Berlin, Berlin, 1999. 

A Lawen, R Traber, D. Geyl. In vitro biosynthesis of [Thr2, Leu5, D-Hiv8, Leu10]-cyclosporin, a cyclosporin-related peptolide, with immunosuppressive activities by a multienzyme polypeptide. J Biol Chem 266 15567-15570, 1991. 

A final example of metabolic pathway engineering is based on polyketide and nonribosomal peptide biosynthesis. Polyketides and nonribosomal peptides are complex natural products with numerous chiral centers, which are of substantial economic benefit as pharmaceuticals. These natural products function as antibiotics [erythromycin A (65), vancomycin (66)], antifungals (rapamycin, amphotericin B), antiparasitics [avermectin Ala (67)], antitumor agents [epothiolone A (68), calicheamicin yj, and immunosuppressants [FK506 (69), cyclosporin A], Because this exponentially growing and intensely researched field has developed, the reader is directed to review articles for additional details.347-359 Also with the potential economic benefit to develop the next blockbuster pharmaceutical, a number of patents and patent applications have been published.360-366... 

Nephrotoxicity is caused by drugs that principally affect the renal hemodynamics of the patient depended on vasodilator prostaglandin biosynthesis or angiotensin converting enzyme (ACE) mediated vasoconstriction drugs causing nephrotoxicity include NSAIDs (fenprofen), ACE inhibitors (captopril, and cyclosporin). 

In vitro and in vivo effects of Leflunomide, Brequinar and Cyclosporine on pyrimidine biosynthesis. Transplant. 

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