Cyclosporins, biosynthesis

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. 

As for the biosynthesis of cyclosporin A, four head-to-tail acetate units are involved in the formation of the 8-carbon chain of olefinic amino acid, and N-methyl groups of cyclosporin A originate from methionine (Kobel et al., 1983 Zocher et al., 1984). The enzyme named cyclosporin synthetase has been shown to be responsible for cyclosporin biosynthesis (Billich and Zocher, 1987 Lawen et al., 1989). This enzyme was subsequently purified and characterized (Lawen and Zocher, 1990). 

In summary, the incorporation of constitutional and foreign amino acids is a hmher chaiacteristic indication for a nonribosomal biosynthetic pathway for cyclosporins. However, exchange of amino acid constituents is limited many substrates tried were not incorporated or acted to suppress in vivo cyclosporin biosynthesis (e.g., DL 3-aminobu tyric acid or S-ethyl-L-methionine). 

The mechanism of cyclosporin biosynthesis is now widely understood, as key enzymes Bmt synthase, alanine racemase. and cyclosporin synthetase are necessary to build up the cyclic undecapeptide. Still more experimental work has to be performed on the biosynthesis of the peptolide SDZ 214 103 to fiilly characterize the enzymes and the pathways involved. 

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... 

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. 

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