Synthetase cyclosporin

Weber, G., Schorgendorfer, K., Schneider-Scherzer, E. and Leitner, E. (1994) The peptide synthetase catalyzing cyclosporine production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame. Current Genetics, 26 (2), 120-125. 

In linear NRPSs a product consisting of amino acids is biosynthesized in an N- to C-terminal manner by the multidomain assembly line with a domain organization of A-PCP-(C-A-PCP) i-TE. The initiation module of a linear NRPS lacks a C domain, while the following modules may include any required additional domains. After formation of the full-length peptide, the product is released from the assembly line by a termination domain. Thus, the number and order of amino acids in the peptide directly coincides with the number and order of synthetase modules. Many NRPs are biosynthesized in this manner, and characterized examples include the penicillin tripeptide precursor -(L-0 -aminoadipyl)-L-cysteinyl-D-valine (ACV, Figure 4 (a)), complestatin, cyclosporin, fengycin, surfactin, and tyrocidine. "... 

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... 

Many NRPs such as cyclosporin, complestatin, actinomycin, and chondramide contain N-methyl amides. M-Methyl transferase (N-MT) domains utilize S-adenosylmethionine (SAM) as a cofactor to catalyze the transfer of the methyl group from SAM to the a-amine of an aminoacyl-S-PCP substrate. The presence of M-methylamides in NRPs is believed to protect the peptide from proteolysis. Interestingly, N-MT domains are incorporated into the A domains of C-A-MT-PCP modules, between two of the core motifs (A8 and A9). MT domains contain three sequence motifs important for catalysis. ° 0-Methyl transferase domains are also found in NRPSs and likewise use the SAM cofactor. For instance, cryptophycin and anabaenopeptilide synthetases contain 0-MT domains for the methylation of tyrosine side chains. These 0-MT domains lack one of the three core motifs described for N-MT domains. ... 

Figure 2 Order and organization of enniatin synthetase and cyclosporin synthetase as deduced from gene sequence and biochemical characterization. Symbols in the adenylateforming modules (black boxes) indicate the corresponding activated amino acids. M stands for A -methyltransferase domain. Condensation domains are represented by white boxes. (A) Top Structure of enniatin synthetase. EA represents the D-Hiv-activating module EB represents the L-valine-activating module D-Ehv is D-2-hydroxyisovaleric acid. Bottom Structural features of the wild-type A -methyltransferase domain M of esynl. The black boxes indicate conserved motifs which can be found within methyltransferases and A -methyltransferase domains of peptide synthetases (see also Fig. 3). The numbers indicate the amino acid position in the sequence of Esyn. (B) Structure of cyclosporin synthetase. Abu = L-a-aminobutyric acid Bmt = (4A)-4-[(E)-2-butenyl]-4-methyl-L-threonine.

H Husi, K Schorgendorfer, G Stempfer, P Taylor, MD Walkinshaw. Prediction of substrate-specific pockets in cyclosporin synthetase. FEBS Lett 414 532-536, 1997. 

A Lawen, R Zocher. Cyclosporin synthetase-the most complex peptide synthesizing multienzyme polypeptide so far described. J Biol Chem 265 11355-11360, 1990. 

G Weber, E Leitner. Disruption of the cyclosporin synthetase gene of Tolypocladium niveum. Curr Genet 26 461-467, 1994. 

E Schneider, E Leitner, G Weber, K Schorgendorfer. Cyclosporin synthetase. European Patent 0578616 [Sandoz Ltd (CH) Sandoz AG (DE)], 1994. 

A Lawen, R Traber. Substrate specificities of cyclosporin synthetase and peptolide SDZ 214-103 synthetase. J Biol Chem 268 20452-20465, 1993. 

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

Although most modules of peptide synthetases show a strict specificity towards natural amino acids, some modules can incorporate alternative amino acids into their products. For example, the enniatin synthetase shows a relatively broad substrate specificity for hydrophobic amino acids (L-Val, L-Leu, L-Ile). Both nonmethylated and methylated versions can be processed to generate de-psipeptides in a cell-free system [202]. Likewise, cyclosporins with a variety of alternative amino acids have also been synthesized from cell-free cyclosporin synthetase preparations [203]. Various enniatins and cyclosporins can also be isolated from fermentation broths [204,205]. 

Velkov T, Lawen A. Mapping and molecular modeling of S- 28. adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase. 

Grieve EM, Hawksworth GM, Simpson JG, Whiting PH. Effect of thromboxane synthetase inhibition and angiotensin converting enzyme inhibition on acute cyclosporin A nephrotoxicity. Biochem Pharmacol 1990 40 2323-2329. 

Grieve EM, Hawksworth GM, 5impson JG, Whiting PH.The reversal of experimental cyclosporin A nephrotoxicity by thromboxane synthetase inhibition. Biochem Pharmacol 1993 45 1351-1354. 

Gladue RP, Newborg MF. The protective effects of the thromboxane synthetase inhibitor Dazmegrel on nephrotoxicity in cyclosporine-treated rats.Transplantation 1991 52 837-841. 

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. 

There are other mechanisms to release NRP intermediates from NRPSs, although TE domains are mostly utilized. For example, cyclosporine A is possibly released and macrocyclized by one unusual N-terminal C domain in its synthetase. Besides cyclosporine A, thaxomin A may also employ the same strategy in its biosynthesis. The another uncommon method to release NRP intermediates is to reduce the final carboxy group with the consumption of NAD(P)H by reduction domain, exemplified by nostocyclopeptide biosynthetic system. Nonetheless, TE-catalyzed macrocyclization is the favorable mechanism for product proteolytic... 

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