THE JOURNAL OP BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclosporin Synthetase

Vol. 265, No. 19, Issue of July 5, pp. 11355-11360,199O Printed in U. S. A.

THE MOST COMPLEX PEPTIDE SYNTHESIZING MULTIENZYME POLYPEPTIDE SO FAR DESCRIBED*

(Received for publication, February 26, 1990)

Alfons Lawen and Rainer Zocher From the Znstitut fiir Biochemie und Molekulare Biologic, Technische Universitht Berlin, OE 2, Franklinstrasse 29, D-1000 Berlin 10, Federal Republic of Germany

Cyclosporin A and its homologues are synthesized by a single multifunctional enzyme from their precursor amino acids. Cyclosporin synthetase is a polypeptide chain with a molecular mass of approximately 800 kDa. In 3% polyacrylamide-sodium dodecyl sulfate gels it shows a single band of approximately 650 kDa, which appears to not be glycosylated. The enzyme could be purified to near-homogeneity in five steps. A 72-fold purification was obtained. All constitutive amino acids of cyclosporins are activated as thioesters via aminoadenylation by the same enzyme. Then N- methylation of the thioester-bound amino acids which are present in methylated form in the cyclosporin mol- ecule takes place, whereby S-adenosyl-L-methionine serves as the methyl group donor. Methyltransferase activity is an integral entity of the enzyme; this could be shown by a photoaffinity labeling method. 4’-Phos- phopantetheine is a prosthetic group of cyclosporin synthetase similar to other peptide and depsipeptide synthetases. Cyclosporin synthetase shows cross-re- actions with monoclonal antibodies directed against enniatin synthetase.

Cyclosporin A (Fig. 1) is a cyclic undecapeptide with antiin- flammatory, immunosuppressive, antifungal, and antiparasi- tic properties (1). It is used in transplantation surgery and in the treatment of autoimmune diseases (2, 3).

Cyclosporin A is produced by the fungus Beauveria nivea (previously designated Trichoderma polysporum, Tolypoclud- ium infkztum, and Tolypocladium niveum) as the main com- ponent of 25 naturally occurring cyclosporins which have substitutions of amino acids in positions 1, 2, 4, 5, 7, and 11 and/or contain unmethylated peptide bonds in positions 1,4, 6, 9, 10, or 11 (4). Beside these naturally occurring cyclo- sporins, some cyclosporins differing in positions 1, 2, and 8 from cyclosporin A could be produced by feeding amino acid precursors to the fungus (5).

The structure of cyclosporin A strongly suggested a nonri- bosomal biosynthesis mechanism (6): three unusual amino acids (Bmt’ in position 1, 2-aminobutyric acid in position 2,

* This work was supported by a grant from Sandoz Ltd., Basel, Switzerland. Parts of this work were presented at the 19th Federation of European Biochemical Societies Meeting, July 2-7, 1989, Rome (Abstr. FR 138). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

1 The abbreviations used are: Bmt, (2$3R,4R,6E)-2-amino-3- hydroxy-4-methyl-6-octenoic acid (= (4R)-4-[(E)-2-butenyll-4- methyl-L-threonine); AdoMet, S-adenosyl-I,-methionine; Hepes, 4- (2-hydroxymethyl)-l-piperazineethanesulfonic acid; Me, N-methyl; MOPS, 3-(N-morpholino)propanesulfonic acid; SDS, sodium dodecyl sulfate; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid.

and D-alanine in position 8), seven N-methylated peptide bonds, and the cyclic structure of the molecule. The latter properties cyclosporin A shares with the depsipeptides ennia- tin and beauvericin which have been shown to be synthesized by large multienzyme complexes from their primary precur- sors (amino and hydroxy acids) under ATP and AdoMet consumption (7-9). Synthesis of depsipeptides involves ami- noadenylation of precursors, binding of the activated precur- sors as thioesters, N-methylation of the corresponding en- zyme-bound amino acids, elongation, and cyclization reac- tions (10, 11).

Previous attempts to characterize the enzyme system re- sponsible for synthesis of cyclosporins first led to the enrich- ment of an enzyme fraction catalyzing the synthesis of the diketopiperazine cycle-(o-Ala-MeLeu), representing a partial sequence (positions 8 and 9) of cyclosporin A (12). Although this preparation was able to activate all constitutive amino acids of cyclosporin A as thioesters via aminoadenylation, total synthesis of cyclosporin A was not observed. Further efforts guided to total in vitro synthesis of several cyclosporins by partially purified cyclosporin synthetase fractions (13) and led recently to the in vitro biosynthesis of cyclosporins not obtainable by fermentation (14).

This paper describes further purification and characteriza- tion of cyclosporin synthetase and confirms that cyclosporin synthetase follows a thiotemplate mechanism (15), which has been shown previously for the biosynthesis of various other peptides and depsipeptides (16).

MATERIALS AND METHODS

Growth of Organisms-B. nivea, strain 7939145, was donated by Sandoz Ltd. (Basel, Switzerland) and cultured as described in Ref. 14. Lactobacillus plantarum, DSM 20 205, was maintained on pancul- ture agar (Difco) plates. Pre- and mainculture were incubated in pantothenate assay medium (Difco) at 37 “C. Preculture was har- vested by centrifugation, washed several times with 0.9% NaCl, and used as inoculum.

Radioisotopes and Chemicals-All radiochemicals were purchased from Amersham Corp. Bmt and cyclosporin A were donated by Dr. R. Traber, Sandoz Ltd. (Basel, Switzerland), MeBmt was donated by Dr. R. M. Wenger, Sandoz Ltd. (Basel, Switzerland). ATP was from Boehringer Mannheim. Partially purified tyrocidine synthetase III and linear gramicidin synthetase were kindly provided by Dr. H. von Diihren (Berlin). Enniatin synthetase was purified as described in Ref. 8. All other chemicals used were from Sigma or Merck (Darm- stadt, West Germany) and were reagent grade.

In Vitro Cyclosporin A Formation-For checking the enzyme activ- ity cyclosporin A was synthesized as described in Ref. 14, but using 0.125 &i of [methyl-‘4C]AdoMet. For specific activity determinations some different substrate concentrations were used: 8 mM MgCl?; 3.5 mM ATP; 0.125 mM Bmt; 0.5 mM of each of the other constituent amino acids of cyclosporin A and 0.35 mM [methyl-‘4C]AdoMet (5.8 Ci/mol). 100 ~1 of enzyme were incubated in a total volume of 120 ~1 at 25 “C for 15 min. The reaction was stopped, and the cyclosporin

11355

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11356 Cyclosporin Synthetase

cna 0’t:

cm C”,

D-AI. Al* MeLOU VII YeLOU

FIG. 1. Structure of cyclosporin A. Abu, 2-aminobutyric acid; Sar, sarcosine.

formed was extracted as described in Ref. 13. TLC analysis was done as described (14).

Thioester Formation-This was measured by incubating 100 ~1 of enzyme in a total volume of 160 pl with 31 mM MgC12, 28 mM ATP, and 0.5 &i of “C-labeled amino acids (specific activities: threonine, 288 mCi/mmol; glycine, 108 mCi/mmol; leucine, 348 mCi/mmol; valine, 285 mCi/mmol; alanine, 171 mCi/mmol; D-alanine, 40 mCi/ mmol) or (in the presence of 156 pM Bmt) 0.25 &i of [nethy[-“C] AdoMet (56 mCi/mmol) for 10 min at 25 “C. The reaction was stopped by addition of 2 ml of 7% trichloroacetic acid. After 30 min on ice the precipitate was collected on membrane filters (ME 25, Schleicher & &hull, Dassel, West Germany). Filters were washed twice with 7% trichloroacetic acid and water each; after drying, the radioactivity was determined.

Enzyme Pt.&cation-Extraction of lyophilized mycelium and pre- cipitations with 0.3% polyethyleneimine and 30-50% saturated (NH&SO, were achieved as described earlier (14). 10 ml of the resuspended (NH&SO, precipitation material were loaded onto a Fractogel HW-55 (F) column (Merck, Darmstadt; 4 x 63 cm) and eluted with buffer B (0.1 M Tris-HCl, pH 7.8; 4 mM EDTA; 4 mM dithiothreitol; 15% (w/v) glycerol) (14). Active fractions were pooled, and 7 ml of the pool were loaded onto a 33-ml gradient from 50-25% (w/v) glycerol in buffer C (0.1 mM Tris-HCl, pH 7.8; 50 mM KCl; 4 mM EDTA; 4 mM dithioerythritol) in a Beckman SW 27 tube. Ultracentrifugation was for 6 h at 10,000 rpm and for 48 h at 22,000 rpm. Gradients were harvested from bottom to top.

For molecular weight determinations a Beckman SW 41 rotor was used; the molecular weight standards used were ribosomal 40 S subunits prepared according to Ref. 17, thyroglobulin, and katalase (Boehringer Mannheim).

Protein was determined by a dye-binding method (18) using bovine serum albumin as standard.

SDS-Gel Electrophoresis-This was done routinely in gradient gels from 15 to 2% polyacrylamide in the Laemmli system (19). For molecular weight estimations 3% polyacrylamide gels in the same system were used. Gels were fixed and stained with Coomassie blue or fixed with 50% methanol, 12% acetic acid, fluorographed with Amplify (Amersham), and autoradiographed using an x-ray film (Konica, Tokyo, Japan, or Amersham (P-Max), Braunschweig, West Germany).

Glycoproteins were stained using Schiffs reagent (Sigma) follow- ing the procedure described by Fairbanks et al. (2.0).

Affinity Labeling of Cyclosporin Synthctuse-To 100 ~1 of a cyclo- sporin synthetase preparation from glycerol gradients were added 5 ~1 of [methyl-3H]AdoMet (5 &i), 10 ~1 of [methyl-YZ]AdoMet (0.25 PCi), 10 ~1 of [carboxyl-‘“C]AdoMet (0.25 &i), or 10 ~1 of [U-Y] ATP (0.5 j&i). The mixtures were irradiated at 4 “C for up to 15 min with a short-wave UV light (254 nm) from a 44-watt mercury lamp from a distance of 2 cm as described previously (21, 22).

Reactions were stopped by adding 1 volume of lysis buffer (0.015 M Tris-HCl, pH 6,s; 8 M urea; 1% @-mercaptoethanol; 1% SDS; 10% glycerol) and incubating the mixture for 5 min at 95 “C. Some reaction mixtures were first incubated with 1 rg of trypsin or protease from Staphylococcus aureus, strain V8 (both from Sigma) for 2 h at 25 “C and stopped thereafter.

Determination of 4’-Phosphopantethein-Cyclosporin synthetase fractions (540 ~1 each) from a glycerol gradient were hydrolyzed with 1 N KOH for 1 h at 100 “C, incubated after adjusting a pH of 8 successively for 2 h at 37 “C and overnight at 4 “C with or for control without 1.3 units of bovine alkaline phosphatase (Sigma P-2276). Pantothenic acid liberated from enzyme-bound 4’-phosphopanteth- eine was determined microbiologically using L. plantarum (DSM 20205) as the test organism as described previously (8, 23).

RESULTS

Purification of Cyclosporin Synthetase-Cyclosporin syn- thetase from B. niuea, strain 7939145, was purified 72-fold. The purification protocol is presented in Table I. At any step of the purification procedure, the enzyme could be stored at -80 “C for over 12 months without loss of activity. Prepara- tion of the crude extract and precipitation with polyethylene- imine and with (NH&SO, were achieved as described in Ref. 14. The redissolved ammonium sulfate precipitation material was separated by gel filtration on a Fractogel HW-55 (F) column; the activity resided in a single peak (Fig. 2).

Due to the high molecular weight of the enzyme, further purification could be achieved by glycerol gradient ultracen- trifugation (Fig. 3). Examination of the different purification steps by SDS-polyacrylamide gradient gel electrophoresis shows that cyclosporin synthetase is the major protein after the ultracentrifugation step (Fig. 4A).

Purification of the cyclosporin synthetase activity was fol- lowed by measuring the cyclosporin A synthesis rate as de- scribed under “Materials and Methods.” Subsequently TLC and autoradiography were performed to confirm the cyclo- sporin A production (Fig. 4B).

TABLE I

Purification of cyciosporin synthetase 25 g lyophilized mycelium were used. For more details see “Mate-

rials and Methods.”

Step Volume Protein Units Specific activity

ml w picokatal femtokatalf w

1. Crude extract 500 2,226 470 211 2. Polyethyleneimine pre- 500 1,722 828 481

cipitation 3. 30-50% (NH&SO4 9 299 505 1,689

precipitation 4. Fractogel HW-55 col- 103 59 988 16,746

umn 5. Glycerol gradients (25- 108 31 345 11,129

50%)

r

FIG. 2. Elution profile of Fractogel HW-55 (F) column. 8 ml of resuspended ammonium sulfate precipitate were loaded onto a Fractogel HW-55 (F) column (4 x 63 cm) and eluted with buffer B. S-ml fractions were collected; protein content (0 - - -0) and cyclo- sporin synthetase activity (A - A) of each fraction were measured.

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Cyclosporin Synthetase 11357

r

1 5 10 15 20

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F ractlon number

Frc. 3. Elution profile of glycerol gradient ultracentrifu- gation. 7 ml of pooled active fractions of Fractogel chromatography were loaded onto a glycerol gradient from 50 to 25% glycerol in buffer C and centrifuged as described under “Materials and Methods.” 2-ml fractions were collected; protein content (0- - -0) and cyclosporin synthetase activity (A-A,) of each fraction were measured.

Interestingly cyclosporin synthetase activity could also be isolated from spores of B. niuea using the extraction procedure described above.

Molecular Mass Determinations-Measurements of the mo- lecular weight of the native cyclosporin synthetase were per- formed by ultracentrifugation in glycerol gradients along with standard proteins. A molecular mass of about 800 kDa was obtained. The apparent molecular weight of denatured en- zyme was determined by SDS-polyacrylamide gel electropho- resis (3% gels); extrapolation of molecular masses of calibra- tion proteins results in a molecular mass between 600 and 700 kDa for cyclosporin synthetase (Fig. 5). When protein fragments yielded from trypsin digestion of cyclosporin syn- thetase were separated in SDS-gradient gels, addition of their molecular masses gave a value of about 750 kDa, which is in good agreement with the findings described above.

Carbohydrate Stain-To check whether cyclosporin synthe- tase is a glycoprotein we used a staining procedure which is sensitive for carbohydrate portions of proteins. However no staining with Schiffs reagent could be observed. This finding together with the fact that cyclosporin synthetase does not bind to concanavalin A-agarose led us to the assumption that cyclosporin synthetase is not a glycoprotein.

Biosynthesis of Cyclosporins-To synthesize cyclosporins in vitro cyclosporin synthetase needs the appropriate amino acids in unmethylated form (including D-alanine or a homo-

A “q-

- - p-r-CySv

f-P 3 f _I

12 34 5

B

FIG. 4. Purification of cyclosporin synthetase. A, cyclosporin synthetase purification was followed by electrophoresis in 15-2% Laemmli polyacrylamide SDS gels. 1 ml of each of the first three purification steps was desalted by passage through PD-10 columns (Pharmacia, Freiburg, West Germany) and prepared for gel electro- phoresis (see “Materials and Methods”). Lane I, 25 ~1 of crude extract; lane 2, 25 ~1 of extract after precipitation with 0.3% polyethylene- imine; lane 3, 5 ~1 of 30-50% saturated (NH,),SO, precipitation; lane 4, 25 ~1 of pooled active fractions of Fractogel HW-55 chromatogra- phy; lane 5, 25 @I of pooled active fractions of glycerol gradient ultracentrifugation. The gel was stained with Coomassie blue; the position of cyclosporin synthetase (CySyn) is indicated. R, 100 11 each of the enzyme preparations from the purification steps men- tioned in A were tested for cyclosporin synthetase activity as de- scribed in Ref. 14. The autoradiogram of the TLC separation of the ethyl acetate extracts is shown. CyA, cyclosporin A.

logue D-amino acid (14); the nonchiral amino acid glycine can also substitute D-alanine),* ATP, Mg’+, and AdoMet as the methyl donor (13, 14). Mg” ions can be substituted by Mn’+ ions. However, the rate of cyclosporin A formation decreases to about 50% compared to the reaction with Mg”. As shown in Ref. 14, the optimal temperature for in uitro cyclosporin A synthesis is 24 “C, the reaction proceeds linearly for at least 15 min under substrate saturating conditions. Cyclosporin A synthesis is inhibited by the reaction products AMP, PP, (not by P,) and S-adenosyl-L-homocysteine, but not by cyclosporin A itself. The reaction proceeds optimally at pH 7.5, measured in Hepes buffer, which is the best buffer for in oitro cyclo- sporin A synthesis, followed by MOPS, Tris, TES, and, at a

’ A. Lawen, J. Dittmann, and R. Traber, unpublished results.

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11358 Cyclosporin Synthetase

kDa

L4 -CySyn

450-

350-

250-

FIN. 5. Molecular mass estimation of denatured cyclosporin synthetase. 25 ~1 of a glycerol gradient enzyme preparation were denatured for gel electrophoresis as described under “Materials and Methods” and separated in a 3% polyacrylamide SDS gel. Extrapo- lation of molecular maaaea of the calibration proteins enniatin syn- thetase (250 kDa), linear gramicidin synthetase (350 kDa), and ty- rocidin synthetase III (450 kDa) results in a molecular mass of 650 kDa for cyclosporin synthetase (CySyn).

great distance (50% of the synthesis rate measured in Hepes), by phosphate buffer.

Activation of Amino Acids and N-Methylation-As it has been reported in Ref. 12 for the cycle-(D-Ala-MeLeu) synthe- sizing enzyme, the cyclosporin synthetase described here cat- alyzes ATP-pyrophosphate exchange reactions dependent on all constitutive amino acids of cyclosporin A in their unmeth- ylated form, whereas the N-methyl amino acids are not acti- vated by the enzyme. Furthermore all amino acids required for cyclosporin C synthesis could be shown to be bound covalently as thioesters to the enzyme (Fig. 6). Cyclosporin C, in which 2aminobutyric acid in position 2 is replaced by threonine (= [Th?]cyclosporin A) was selected for these experiments because 2-aminobutyric acid was not commer- cially available in a W-labeled form. The same holds true for Bmt; covalent binding of this compound to the enzyme was measured indirectly by formation of [N-methyl-‘*C]MeBmt using S-adenosyl[methyl-‘“Clmethionine and unlabeled Bmt. With all “C-labeled amino acids used in Fig. 6, it was also possible to label cyclosporin synthetase specifically as ana- lyzed in polyacrylamide gradient gels (not shown).

Photoaffinity Labeling of Cyclosporin Synthetase-We were interested to clarify, whether the methyltransferase activ- iti is an integral part of the cyclosporin synthetase mole- cule or whether it is an associated but different enzyme. For this purpose we used a method for site-specific affinity label- ing of methyltransferases (21), which has been previously helpful to demonstrate that the methyltransferase activity of enniatin synthetase is an integral part of the enzyme (22). By irradiation with short-wave UV light in the presence of AdoMet labeled in the methyl group various methyltransfer-

Fraction No.

FIG. 6. Thioester-bound amino acids of cyclosporin C. Indi- vidual fractions of a glycerol gradient ultracentrifugation were tested for their capacity to bind the constitutive amino acids of cyclosporin C as thioesters. The W-labeled amino acids were incubated together with ATP, MgCl?, and cyclosporin synthetase as described under “Materials and Methods.” The protein was precipitated with 7% trichloroacetic acid, and the protein-bound radioactivity was meas- ured. Values were corrected with results from incubations without ATP resp. Bmt. The peak fraction (when measured for in uitro synthesis of cyclosporin A) was fraction 8.

ases could be labeled covalently. Like these enzymes cyclo- sporin synthetase, too, was labeled when irradiated in the presence of [methyl-“‘C]AdoMet or [methyl-“H]AdoMet (Fig. 7). Irradiation of the enzyme in the presence of [carboxyl-‘4C] AdoMet or [U-‘%]ATP did not give any labeling (not shown). When the affinity-labeled enzyme was digested either with trypsin or with S. aureus V8 protease, three radiolabeled protein bands arose (Fig. 7), suggesting the presence of more than one methyltransferase activity per cyclosporin synthe- tase molecule, probably three; work is in progress to determine the exact stoichiometry.

Immunological Examinations-At the ultracentrifugation stage of purification only the major protein band and some minor bands of the preparation running just a little faster in the gel show a positive reaction with a polyclonal rabbit antiserum specifically directed against cyclosporin synthe- tase.” These minor bands seem to represent degradation prod- ucts of the enzyme, for they show a behavior very similar to cyclosporin synthetase. It is not yet clear whether they origi- nate proteolytically or mechanically from degradation; never- theless their concentration does not increase when prepara- tions are standing at 4 “C, indicating the absence of proteases in the preparations.

The cyclosporin synthetase band cross-reacts in immuno- blots with a polyclonal antiserum directed against enniatin synthetase as well as with the monoclonal antibodies against enniatin synthetase described in Ref. 24. With the latter antibodies, the strongest reactions could be detected with monoclonal antibodies 21.1 and 25.91, which inhibit the thioester formation with valine and recognize the denatured form of enniatin synthetase.

Furthermore, cyclosporin synthetase preparations show a very significant cross-reaction with a polyclonal antibody preparation directed specifically against pantetheine in en- zyme-linked immunosorbent assay,4 suggesting the presence of 4’-phosphopantetheine as a prosthetic group similar to a

’ A. Lawen, K. Hoffmann, and R. Zocher, unpublished results. 4 A. Lawen, A. Billich, and R. Zocher, unpublished results.

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Cyclosporin Synthetase

A B kDa b v

-4 250- 205-

G

I A?--* -* - -

66-

45- - L,

29-- I,

I 1112 3 723

1

FIG. 7. Photoaffinity labeling of the methyltransferase en- tity of cyclosporin synthetase. 100 ~1 of a cyclosporin synthetase preparation from an ultracentrifugation were irradiated at 254 nm with [merhy-“C]AdoMet as described under “Materials and Meth- ods” (lane I). After irradiation the enzyme was digested either with tr.ypsin (lane 2) or with S. aureus V8 protease (lane 3). The protein was separated by polyacrylamide gel electrophoresis in a 15-2% Laemmli gel and stained with Coomassie blue (A) or autoradi- ographed after fluorography (R). The molecular masses of standard proteins are indicated (A).

number of other peptide and depsipeptide synthetases. Presence of 4’.Phosphopantetheine in Cyclosporin Synthe-

tase-To confirm the assumption that 4’-phosphopantetheine forms part of cyclosporin synthetase, we performed a micro- biological assay with Lactobacillu.s as a test organism. Frac- tions from the glycerol gradient ultracentrifugation step were analyzed in order to determine their 4’-phosphopantetheine content. As shown in Fig. 8 the synthetic activity of cyclo- sporin synthetase comigrates with panthotenate in the gra- dient. The fact, that most of the panthotenate was released after alkaline phosphatase treatment proves that it is present as 4’-phosphopantetheine in the enzyme. In addition the SDS-polyacrylamide gel electrophoresis separation shows the typical band of cyclosporin synthetase comigrating with pan- tothenate release and synthetic activity (Fig. 8). Further evidence for the presence of 4’-phosphopantetheine in cyclo- sporin synthetase was obtained from specific labeling of the enzyme by in vivo feeding of tritiated /3-alanine, which was analyzed by polyacrylamide gel electrophoresis and adjacent autoradiography (not shown).

DISCUSSION

The first attempts to establish the cell-free synthesis of cyclosporin were not successful, but led to an enzyme enrich- ment actively synthesizing the diketopiperazine cycle-(D-Ala- MeLeu) (12), which represents a partial sequence of cyclo- sporin A. Change of the cyclosporin producer strain and the buffer for enzyme preparation (Tris buffer instead of phos- phate, glycerol content) resulted in successful in uitro synthe- sis of cyclosporin (13). From our results Hepes and Tris are appropriate buffer systems for the enzyme in contrast to the

r kDa

205 -'-

116 -._ 91 ---

66 -*

L5 -

29 -\

1 5 10 15

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Fraction number

Fm. 8. Release of 4’-phosphopantetheine from cyclosporin synthetase. Cyclosporin synthetase fractions from a glycerol gra- dient were hydrolyzed with 1 N KOH, incubated without (0) or with (0) alkaline phosphatase and tested in an microbiological assay with L. plantarum as described under “Materials and Methods.” The pantothenate release comigrates with the in vitro synthesis of cycle- sporin A (X). The Coomassie blue-stained Laemmli gel (15-2% poly- acrylamide) separation of the gradient fractions is shown above; molecular masses of the standard proteins are indicated.

previously used phosphate buffer; the presence of glycerol in the buffer is necessary as a stabilizer. We think that in the absence of glycerol some conformational changes of the en- zyme take place which lead to the loss of its ability to produce cyclosporins. The main reaction product of such “inactive” enzyme preparations is the diketopiperazine cyclo-(D-Ala- MeLeu).” Therefore it seems obvious that our previous prep- arations described in Ref. 12 contained intact but “inactive,” probably conformationally changed, cyclosporin synthetase polypeptide chains.

Like enniatin synthetase (8, 22) which can be considered as a model system for other N-methylating peptide synthe- tases cyclosporin synthetase accepts only the unmethylated precursor amino acids of cyclosporins which are methylated while bound to the enzyme as thioesters as previously shown (Ref. 12). The methyltransferase(s) responsible for these N- methylations is integral part of the enzyme as could be shown by the affinity-labeling experiments with [methyl-“‘Cl AdoMet.

It is interesting that all peptide and depsipeptide synthe- tases from fungi (e.g. enniatin (8), beauvericin (ll), b-(L-cy- aminoadipyl)-L-cysteinyl-D-valine (26), ergot peptide lactam,” and cyclosporin synthetase) do not exhibit subunit structure. They consist of single polypeptide chains of molecular masses between 250 and 800 kDa, which harbor all catalytic activities necessary for peptide formation. Such enzymes are designated as “multienzyme polypeptides” in the nomenclature according to NC-IUB (27), in contrast to the “multienzyme complexes” from prokaryotes which consist of subunits (e.g. gramicidin, tyrocidin, bacitracin synthetase, for review see Ref. 16).

‘J. Dittmann, R. Zocher, and A. Lawen, unpublished results. ” N. Quandt and U. Keller, personal communication.

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11360 Cyclosporin Synthetase

Experiments to determine the exact number of N-methyl- transferase(s) and 4’-phosphopantheteine residues per mole of cyclosporin synthetase have been hampered by two diffi- culties. First, to measure exactly the absolute protein content of our preparations, because the dye-binding method we used is related to the calibration protein (bovine serum albumin in our case). Attempts to determine the protein amount gravi- metrically were not successful, as we believe, due to different glycerol quantities remaining associated to the enzyme. Sec- ond, we never know the exact quantity of inactivated enzyme in our preparations. As can be seen from Table I during the last purification step, a considerable loss of specific activity is observed.

The molecular mass of cyclosporin synthetase has been determined to be between 650 and 800 kDa. In spite of this high value, we were not able to dissociate the enzyme into subunits; neither with urea nor with detergents like SDS nor with P-mercaptoethanol.

This high molecular mass is not astonishing if one realizes that there are (in the case of cyclosporin A) 7 amino acids which have to be N-methylated and in total 11 amino acids which have to be activated and combined. The overall reaction of cyclosporin synthesis can be divided in at least 40 partial reaction steps: 11 aminoadenylation reactions, 11 transthiol- ation reactions, 7 N-methylation reactions, 10 elongation reactions, and the final cyclization reaction); possible other transthiolation reactions from one thiol group to another are not included in this calculation, The measured molecular mass is in good agreement with a theory of Lipmann and co-workers (28), which requires a protein domain of 70 kDa for each activation site in a peptide synthetase; so, in the light of this assumption one would expect a molecular mass of 770 kDa for cyclosporin synthetase.

In summary, cyclosporin synthetase appears to be the larg- est and most complex enzymatically active multienzyme poly- peptide chain so far described and is a further example of a N-methylating peptide synthetase from eukaryotes.

Acknowledgments-We are grateful to Drs. T. Payne, A. Billich, E. Schneider-Scherzer, and H. Kleinkauf for valuable discussions and R. Stepanek for technical assistance.

REFERENCES

1. Borel, J. F. (1986) Prog. Allergy 38.9-18 2. Kahan, B. D. (ed) (1984) Cyclosporin: Biological Activity and

Clinical Applications, Grune & Straton Inc., Orlando, FL 3. Schindler, R. (ed) (1985) Cyclosporin in Autoimmune Diseases,

Springer Verlag, Berlin 4. Traher, R., Hofmann, H., Loosli, H. R., Ponelle, M., and von

Warthurg, A. (1987) Helu. Chim. Acta ‘70, 13-36 5. Traher, R., Hofmann, H., and Kobel, H. (1989) J. Antibiot. 42,

591-597 6. Zocher, R., Madry, N., Peeters, H., and Kleinkauf, H. (1984)

Phytochemistry 23,549-551 7. Zocher, R., and Kleinkauf, H. (1978) Biochem. Biophys. Res.

Commun. 81, 1162-1167 8. Zocher, R., Keller, U., and Kleinkauf, H. (1982) Biochemistry 21,

43-48 9. Peeters, H., Zocher, R., Madry, N., Oelrich, P. B., Kleinkauf, H.,

and Kraepelin, G. (1983) J. Antibiot. 36,1762-1766 10. Zocher, R., Keller, U., and Kleinkauf, H. (1983) Biochem. Bio-

phys. Res. Commun. 110,292-299 11. Peeters, H., Zocher, R., and Kleinkauf, H. (1988) J. Antibiot. 41,

352-359 12. Zocher, R., Nihira, T., Paul, E., Madry, N., Peeters, H., Kleinkauf,

H., and Keller, U. (1986) Biochemistry 25,550-553 13. Billich, A., and Zocher, R. (1987) J. Biol. Chem. 262, 17258-

17259 14. Lawen, A., Traber, R., Geyl, D., Zocher, R., and Kleinkauf, H.

(1989) J. Antibiot. 42, 1283-1289 15. Lipmann, F. (1971) Science 173,875-884 16. Kleinkauf, H., and van Dohren, H. (1987) Annu. Reu. Microbial.

41,259-289 17. Blobel, G., and Sabatini, D. (1971) Proc. N&l. Acad. Sci. U. S. A.

68,390-394 18. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 19. Laemmli, U. K. (1970) Nature 227,680-685 20. Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Bio-

chemistry 10,2606-2617 21. Hurst, J. H., Billingsley, M. L., and Lovenberg, W. (1984)

Biochem. Biophys. Res. Commun. 122,499-508 22. Billich, A., and Zocher, R. (1987) Biochemistry 26,8417-8423 23. Pugh, E. L., and Wakil, J. (1965) J. Biol. Chem. 240,4727-4733 24. Billich, A., Zocher, R., Kleinkauf, H., Braun, D. G., Lavanchy,

D., and Hochkeppel, H.-K. (1987) Biol. Chem. Hoppe-Seyler 368,521-529

25. Deleted in nroof 26. van Liempt, H., von Dohren, H., and Kleinkauf, H. (1989) J.

Biol. Chem. 264.3680-3684 27. Nomenclature Committee of the International Union of Bio-

chemistry (NC-IUB) (1989) Eur. J. Biochem. 185,485-486 28. Lee, S. G., Roskoski, R., Jr., Bauer, K., and Lipmann, F. (1973)

Biochemistry 12,398-405

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A Lawen and R Zocherpolypeptide so far described.

Cyclosporin synthetase. The most complex peptide synthesizing multienzyme

1990, 265:11355-11360.J. Biol. Chem. 

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