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Biochimie (1992) 74, 511-516 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

Enzymatic biosynthesis of cyclosporin A and analogues* A L a w e n 1, J Dittmann], B Schmidt 2, D Riesner2, H

Kleinkaufl

ITechnical University Berlin, Institute of Biochemistry and Molecular Biology, Franklinstr 29, D-(W)-IO00 Berlin 10; "-Institute of Physical Biology. Heinrich-Heine University Diisseldorf, Universitiitsstr 1, D-(W)-4000 Diisseldorf, Germany

(Received 26 August 1991; accepted 15 November 1991)

- - The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyciosporin synthetase. This muir,enzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately l 400 kDa by two different methods: 1), by 3% SDS-PAGE using the related muir,enzymes ACV synthetase and gramicidin $ synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsC! density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid]]CyA, dihydro-CyA, [L-norvaline2.5, N-methylL-norvaline:t ]CyA, lL-allo-isoleucines, N-methyl-L-allo-isoleucine:llCyA, [D-2-aminobutyric acidS]CyA, l~-chloro-D-alanineS]CyA and some related compounds could be established• By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucineS.1°, D-2-hydroxyisovaleric acidS]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass s.pectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are :mmunosuppressive. Summary

cyclosporin

A / biosynthesis

/ new cyclosporins

/ muitifunctionai

Introduction Cyclosporin A (CyA; fig 1) is a cyclic undecapeptide with a molecular mass of 1 202 Da which exerts antifungal, ant,parasitic, anti-inflammatory and immunosuppressive activity [1]. It is commonly used in human transplantation surgery and in the treatment of autoimmune diseases [2, 3]. CyA is the main component to date, of the 25 naturally occurring cyclosporins which have been described as being produced by the fungus Beauveria nivea (previously designated as Tolypocladium inflaturn). All cyclosporins have the basic structure shown in figure 1; substitutions of amino acids have been observed in positiens 1, 2, 4, 5, 7 and 11, and unmethylated peptide bonds have been reported in positions 1, 4, 6, 9, 10 and 11 [4]. By feeding amino acid precursors to the fungus, several cyclosporins with substitutions in positions 1, 2, and 8 have been obtained [5].

/ peptolide SDZ 214-103

polypeptide

In 1984, Wenger described the first total chemical synthesis of cyclosporin [6], which led to the synthesis of several hundred CyA analogues [7, 8]. Never-

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Abbreviations: ACV, 5-(L-a-Aminoadipyl)-L-cysteinyl-D-valine; CyA, cyclosporin A; PAGE, polyacrylamide gel electrophoresis; Me, N-methyl; PPIase, peptidyi-prolyl cis-trans isomerase; SDS, sodium dodecyl sulfate.

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Fig 1. Structure of CyA. Abu, 2-aminobutyric acid; Bmt, (4R)-4-[(E)-2-butenyl]4-methyl-L-threonine; Sat, sarcosine.

512 fluares¢lnce

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Fig 2. Molecular mass estimation of cyclosporin synthetase by density gradient centrifugation [28]. Cyclosporin synthetase purified by glycerol gradient ultracentrifugation [18] has been labeled with 4'-(((iodoacetyl)amino)methyl)fluorescein, 8.8 gg of this enzyme preparation (end concentration 25 ~tg/ml) were analyzed by CsCI density gradient centrifugation at 40 000 rpm in an analytical ultracentrifuge equipped with a fluorescence detection system. The profile shows the distribution of fluorescence intensity monitored after 9 h. Open circles designate the best fit to the Gaussian distribution equation: (r-rx)2 1 f (r)

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r mrepresents the meniscus, r b the bottom of the measuring cell, r~ the radial position of the protein band, o the bandwidth (Schmidt Bet al, manuscript in preparation). theless, CyA possesses the highest immunosuppressive activity of all cyclosporins. A cellular cyclosporin binding protein, a cytosolic 17.7 kDa-protein named 'cyclophilin' has been described [9, 10]. This protein was shown to be identical to the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) [ 11] by amino acid sequence comparison [ 12, 13]. PPlase activity is inhibited by cyclosporin A [12, 13]. Most cyclosporin analogues tested so far show a good correlation between cyclophilin binding, inhibition of PPIase activity and immunosuppressive activity [7, 14]; nevertheless some discrepancies exist which are yet not understood [ 15].

responsible for cyclosporin synthesis has been characterized as one single multienzyme polypeptide [18]. Its molecular mass was originally determined at at least 650-800 kDa [18]. Recent data for ACV synthetase, the first enzyme of the penicillin biosynthesis, show that its molecular mass has been dramatically underestimated (230 kDa instead of 425 kDa) [ 19-21 ]. Using this enzyme as reference, we obtained an apparent molecular mass of 1.5 MDa for cyclosporin synthetase in 3% SDS-PAGE (Lawen A, unpublished observations). The value of 570 kDa obtained for gramicidin S synthetase 2 in the same experiment is in good agreement with recent data from laser desorption mass spectroscopy experiments (556 + 5 kDa; Vestal M, von DOhren H, unpublished observations). CsCI density gradient experiments of fluorescence-labeled cyclosporin synthetase were carried out in an analytical ultracentrifuge with a fluorescence detection system [22]. From the band-width of the macromolecular distribution a molecular mass of about 1.4 MDa was determined (fig 2). Like many other peptide and depsipeptide synthetases, cyclosporin synthetase contains 4'-phosphopantetheine as the prosthetic group. The enzyme appears not to be glycosylated [ 18]; its activity is not affected by treatment with ribonuclease A (Lawen A, unpublished observations). Cyclosporin synthetase activates all constituent amino acids of CyA in their unmethylated form as adenylates (measured by amino acid dependent ATP/pyrophosphate exchange) and binds these covalently as thioesters. At this stage, Nmethylation of the amino acids 1, 3, 4, 5, 9, 10, and 11 takes place, followed by peptide bond formation and cyclization. Thus, at least 40 reaction steps are carried out by one polypeptide chain [18]. Free CyA has been described to possess in several solvents a cis-amide bond between the methylleucines in positions 9 and 10 ([8] and references therein). It is not clear to date however, whether cyclosporin synthetase produces this cis-amide bond or not. We were able to show that in vitro CyA synthesis is not influenced by the presence of various concentrations of pig kidney cyclophilin (a generous gift from C.H3

Cyclosporin synthetase Cyclosporin A has been shown to be synthesized by a nonribosomal mechanism [16, 17]. The enzyme system

Fig 3. Structure of the diketopiperazine cyclo-(D-AlaMeLeu).

513 Dr G Fischer, Halle) in the assay mixture (Lawen A, unpublished observations). This result can be interpreted as an indication that the cis-amide bond is not enzymatically catalyzed, but originates spontaneously in some solvents.

Front

N-Methyitransferase activity S-Adenosyl-L-methionine acts as a methyl donor in cyclosporin biosynthesis. The methyltransferase activity(ies) of cyclosporin synthetase is similar to the previously described enniatin synthetase [23] an integral part of the enzyme as demonstrated by using a photolabeling method for methyltransferases [18]. The in vitro synthesis of an unmethylated CyA is not possible. Nevertheless with cyclosporin synthetase isolated from a blocked mutant of B nivea which synthesizes the diketopiperazine cyclo-(v-Ala-MeLeu) (fig 3) instead of CyA, an unmethylated product could be obtained (fig 4). As in the case of enniatin synthetase, the unmethylated product is synthesized more slowly than the methylated one [24]. On account of the synthesis of the unmethylated product cyclosporin synthetase can be considered as a peptide synthetase combined with a methyltransferase(s) unit(s).

Biosynthetic mechanism The linemr peptides D-Ala-MeLeu, D-Ala-MeLeu-MeLeu, D-Ala-MeLeu-MeLeu-MeVal and D-Ala-MeLeuMeLeu-MeVal-MeBmt-Abu-Sar-MeLeu-Val were isolated after liberation from the enzyme by treatment with performic acid, indicating that they were bound as thioesters to the enzyme. All these peptides start with D-AIa (position 8) and represent partial sequences of CyA (Dittmann J, Wenger R, Lawen A, unpublished observations).

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In vitro synthesis of cyciosporins

With a partially purified cyclosporin synthetase preparation CyA was obtained together with homologues which either occur naturally or had been obtained by precursor feeding experiments [ 17]. Recently it became possible to synthesize in vitro a number of new cyclosporins which were not obtainable in vivo [25]. These experiments were carded out by incubating at suboptimal temperature for seven days partially purified enzyme preparations from the high-producer strain of B nivea 7939/45 together with all the necessary amino acids in their unmethylated form (including one o-amino acid), ATE MgCl2 and S-adenosyl-Lmethionine. Suboptimal temperature was chosen in order to stabilize the enzyme and to minimize S-ade-

Fig 4. Enzymatic synthesis of cyclo-(o-Ala-Leu). The enzyme fractions were incubated for 2 h at 25°C with [J4C]Leu, o-Ala and with (1) or without (2) S-adenosyl-Lmethionine. The left arrow indicates the position of cyclo(o-Ala-Me-Leu), whereas the right arrow shows the position of cyclo-(D-Ala-Leu) (Dittmann J, Lawen A, unpublished observations).

nosyl-L-methionine degradation. The cyclosporins were extracted and purified chromatographically. Table I summarizes chromatographic data of some of

514 the cyt losporins identified as products of in vitro synthesis experiments. As is shown for ID-Abua]CyA in figure 5, this method is helpfd in synthesizing new cyclospofins not obtainable in vivo. Experiments to obtain [o-AbuS]CyA by feeding D-aminobutyric acid to the fungus resulted in stimulation of CyA production, probably due to the action of a racemase, whereas the new cyclosporin could be obtained in sufficient amounts Table I. Chromatographic data of enzymatically formed cyclosporins. Cyclosporins were separated on silica gel HPTLC plates. Solvents were water-saturated EtOAc (I), EtOAc-MeOH-H~O, 100:5:5 (in volume) (II) or diisoprop.ylether-CHCirMeOH, 6:3:1 (in volume) (III). The running span was 2 x 10 cm (I) or 10 cm (II and III). HPLC was performed as described in [25]. MeAhdo, N-methyl-(+)-2amino-3-hydroxy-4,4-dimethyloctanoic acid; AllylGly, allylglycine; MeCyclohexylAla, N-methyl-L-~-cyclohexylalanine. Cyclosporin

TLC HPLC Rfvalue a-value solvent ! solvent H solvent !Ii

CyA

0.37

0.44

0.42

10.00

CyO = [MeLeul,Nva2]CyA

0.65

0.59

0.68

13.95

Dihydro-CyA [MeAhdo~]CyA

0.40 0.43

0.47 0.48

0.49 0.55

12.18 13.74

[MeCyclohexylalaqCyA 0.48

0.54

0.57

19.79

[Allylgly2]CyA

0.53

0.55

0.48

10.14

CyQ = [Vala]CyA

0.19

0.28

0.20

4.84

CyM = [Nva2.5]CyA 0.53

0.57

0.58

13.61

[NvaZ.S,MeNvall]CyA

0.56

0.61

0.58

14.34

[NvaS,MeNvalqCyA

0.38

0.46

0.45

10.20

[alleS,aMellell]CyA

0.55

0.57

0.53

15.77

[alleSa qCyA t )

0.30

0.42

0.41

10.67

CyU = [Leu6]CyA

0.45

0.48

0.52

8.92

CyV = [Abu7]CyA

0.48

0.53

0.49

11.97

[o-Abua]CyA

0.43

0.47

0.51

13.09

[~-chloro-D-AlaS]CyA

0.49

0.54

0.47

I 1.73

Table II. Immunosuppressive activity of new cyclosporins. Cyclosporin

Biosynthesis

Activity

Cyclosporin A (Sandimmun)

Natural

+++

Cyclosporin A

Enzymatic

+++

[Nva2.5,MeNvalqCyA

Enzymatic

++

[NvaS,MeNvalqCyA

Enzymatic

++(+)

[alleS,aMelle It ]CyA

Enzymatic

++

[alleS,II]CyA

Enzymatic

+

[D-Abus]CyA

Enzymatic

++

+++ = Strong immunosuppressive activity; ++ = moderate activity; + = weak activity (from [25]).

enzymaticaUy. Since no references exist, for the new cyclosporins, it was necessary to produce sufficient quantities for structural proof. Preliminary structural proof was obtained by fast atom bombardment mass spectrometry and the result is shown for [o-AbuS]CyA in figure 5b. A second criterion to confirm a cyclosporin structure is the immunosuppressive activity of this class of cyclopeptides. As can be seen in table II all new cyclosporins so far synthesized in vitro exert immunosuppressive activity in in vitro assays.

Peptolide SDZ 214-103 synthetase In 1988, an account of the structure of a new cyclic peptolide (SDZ 214-103) produced by the fungus Cylindrotrichum Bonorden was published [26], which exerts similar biological activities to CyA. Peptolide SDZ 214-103 (fig 6) is closely related to CyA and has as main structural difference a o-2-hydroxyisovaleric acid in ester linkage at position 8 instead of o-alanine in the cyclosporins. According to the conventional nomenclature, it can be designated as [L-threonine2, Lleucine5.10, D-2-hydroxyisovaleric acidS]CyA. We were able to prepare an enzyme fraction from crude extracts of the mycelium of Cylindrotrichum Bonorden, which is capable of synthesizing peptolide SDZ 214-103 in vitro. Cyclosporin A is no reaction product of the peptolide synthetase, and cyclosporin synthetase does not synthesize peptolide SDZ 214-103 in vitro [27]. Peptolide 214-103 synthetase is clearly distinct from cyclosporin synthetase, however, both enzymes have large size in common and as far as it has been studied, the principal biosynthetic mechanism.

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Conclusions To our knowledge the synthetases described in this communication are the largest enzymatically active polypeptides. Each of them catalyzes at least about

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40 reaction steps; their molecular masses have been estimated to be about 1.4 MDa. Using these enzymes in an in vitro .~ystem a lot of new analogues of cyclosporin A became available. All these new cyclosporins exert remarkable immunosuppressive activity in vitro. As such, they are good candidates to further study the correlations between immunosuppressive activity, cyclophilin binding and PPlase activity. Our in vitro system should be a good tool to synthesize and characterize further cyclospofins, which are probably helpful in learning more about the mechanism of immunosuppression.

MeLeu

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17 18 19

20

21

22

23 24

25 26 27

28

A: partial purification and properties of a multifunctional enzyme from Tolypocladium inflatum. Biocheinistry 25, 550-553 BillichA, Zocher R (1987) Enzymatic synthesisof cyclosporin A. J Biol Chem 262, 17258-17259 Lawen A, Zocher R (1990) Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described. J Biol Chem 265, 11355-11360 van Liempt H, von D/ihren H, Kleinkauf H (1989) &(L-~tAminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is multifunctional peptide synthetase, l Biol Chem 264, 3680-3684 Smith DJ, Earl AJ, Turner G (1990) The multifunctional peptide synthetase performing the first of penicillin biosynthesis in Penicillium chrysogenum is a 421 073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J 9, 2743-2750 Dfez B, Guti6rrez S, Barredo JL, van Solingen P, van der Voort LHM, Martfn JF (1990) The cluster of penicillin biosynthetic genes: identification and characterization of the pcbAB gene encoding the ot-aminoadipyl-cysteinylvaline synthetase and linkage to the pcbC and penDE genes. J Biol Chem 565, 16358-16365 Schmidt B, Rappold W, Rosenbaum V, Fischer R, Riesner D (1990) A fluorescence detection system for the analytical ultracentrifuge and its application to proteins, nucleic acids and viruses. Colloid Polym Sci 268, 45-54 Billich A, Zocher R (1987) N-Methyltransferase function of the multifunctional enzyme enniatin synthetase. Biochemistry 26, 8417-8423 Dittmann J, Lawen A, Zocher R, Kleinkauf H (1990) Isolation and partial characterization of cyclosporin synthetase from a cyclosporin non-producing mutant of Beauveria nivea. Biol Chem Hoppe-Seyler 371,829-834 Lawen A, Traber R, Geyl D, Zocher R, Kleinkauf H (1989) Cell-free biosynthesis of new cyclosporins. J Antibiot 42, 1283-1289 Dreyfuss MM, Schreier MH, Tscherter H, Wenger R (1988) Cyclic peptolides. Eur Pat Appl 0 296 123 A2 Lawen A, Traber R, Geyl D (1991) In vitro biosynthesis of [Thr2,LeuS,D-HivS,Leul0]cyclosporin, a cyclosporin-related peptolide with immunosupprcssive activity by a multienzyme polypeptide. J Biol Chem 266, 15567-15570 Schmid CW, Hearst JE (1969) Molecular weight of homogenous coli phages DNA's from density gradient sedimentation equilibrium. J Mol Bio144, 143-160