Biosynthesis of malformin in washed cells of Aspergillus niger

614

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95386

B I O S Y N T H E S I S OF MALFORMIN IN W A S H E D CELLS OF

ASPERGILLUS

NIGER

M U N E H I K O Y U K I O K A * AND T H E O D O R E W I N N I C K

Department o/ Biochemistry and Biophysics University o[ Hawaii, Honolulu, Hawaii (U.S.A.) (Received August I6th, 1965)

SUMMARY

Washed mycelium of Aspergillus niger, incubated in buffer solution, was found to incorporate suitable 14C-labeled amino acids efficiently into the cyclic peptide, malformin, as well as into protein. Optimum conditions were developed for the assay of this process. Evidence for the specificity of the biosynthesis was that only component amino acids of the malformin molecule were utilized. The chemical identity of the isolated malformin fraction was confirmed b y paper chromatography and by altered paper electrophoresis following oxidation with performic acid.

INTRODUCTION

Current advances in our understanding of the genetic code and the mechanism of protein biosynthesis have stimulated studies on the biogenesis of diverse antibiotic polypeptides of microbial origin 1. Gramicidin S and the tyrocidines of Bacillus brevis have received considerable attention. The experiments of EIKHOM AND LALAND 2, YUKIOKA et al. 3, and MACH AND TATUM4, suggest that these cyclic decapeptides are synthesized b y a route which differs from that for bacterial proteins. On the other hand, HALL et al. ~, have culminated a series of investigations with the demonstration that a specific m R N A codes for gramicidin S synthesis in the presence of ribosomes and soluble components, all derived from B. brevis extracts. Recent experiments in our laboratory have shown that the metabolism of different strains of B. brevis cells is unusually sensitive to conditions of cultivation, and that in certain instances, a limited degree of protein and polypeptide biosynthesis occurs, which is resistant to chloramphenicol, puromycin, or ribonuclease (EC 2.7.7.16). Similar observations are reported by SPAEREN, FROHOLMAND LALAND6. It m a y be that the normal transfer and messenger RNA's become inactive in such cases, due to their destruction b y ribonuclease, or b y combination with nucleotides. In view of this acute interest in the mechanism of polypeptide formation, it * On leave of absence from the Osaka City University Medical School, Osaka, Japan.

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MALFORMIN SYNTHESIS IN ASPERGILLUS EELLS

615

seemed pertinent to extend such studies to additional families of cyclic peptides. Of the many varieties found in lower organisms, one of the most attractive is the malformin group of peptides, which occurs abundantly in cultures of Aspergillus niger. The principal component, malformin A, has been characterized by ANZAI AND CURTIS7 as cyclo-L-isoleucyl-n-cystenyl-L-valyl-D-cystenyl-D-leucyl. This pentapeptide is unique in having the smallest size of any hitherto described natural cyclic peptide, as well as the highest proportion of D-amino acids. The presence of an S-S bridge lends added interest to the structure. For these reasons, the elucidation of the pathway of formation of the malformins offers an intriguing challenge. As a first stage in undertaking this problem, the present paper is concerned with the establishment of optimum conditions for the assay of malformin synthesis in whole ceils taken from Aspergillus cultures. Several types of information have been obtained which should be useful in subsequent studies of the biosynthetic mechanism in cell-free extracts of this organism.

EXPERIMENTAL

Radioactive amino acids These were all [i-14C]labeled. The suppliers and approximate specific activities (mC/mM) were as follows: New England Nuclear Corporation: L-Proline, 200; DL-cystine, 3; DL-leucine, 23; D-leucine, 25; DL-valine, 6; DL-phenylalanine, 6; L-histidine, IO; Calbiochem Company: L-isoleucine, 8; L-alanine, 8; L-tryptophan, 7; L-glutamic acid, 7; Schwartz Bioresearch, Inc.: L-leucine, IO.

A. niger culture Strain 58-883 of the organism was cultivated by the method of TAKAHASHI AND CURTIS8, except that the cells were grown in rotatory shaking culture (2o0

rev./min) in 500 ml flasks containing IOO ml of medium, instead of using the customary aeration procedure in fermentation jars. Unless otherwise stated, the cells were harvested after about 55 h of cultivation at 25-26 °. The yield for these conditions corresponded to approx. 15-16 g of (dry) mycelium/1. The mycelium was collected by filtration, and then washed twice with 500 ml of 0.05 M potassium phosphate buffer (pH 7.5) per original cultivation flask.

Assay o/ mal/ormin biosynthesis To each of a series of 25 ml erlenmeyer flasks was added 4 ml of a suspension of washed mycelium (generally representing 25 mg dry weight) in 0.05 M phosphate buffer (pH 7.5), containing 1 % glucose. Each flask also contained I.oF, C (unless otherwise indicated) of a specified radioactive amino acid, in I ml of phosphate buffer. The flasks were generally incubated with shaking for 6 h at 37 °. The reaction was terminated by transferring the contents of each vessel to a suction filter. The mycelium was then washed with 50 ml of 0.05 M phosphate buffer. The combined filtrate and washings from each experiment were subjected to the earlier stages of the procedure of TAKAHASHIAND CURTIS8, for the isolation of malBiochim. Biophys. Acta, 119 (1966) 614-623

616

M. YUOKIOKA, T. WlNNICK

formin. These operations were adapted to a small scale, as follows: To the above solution (about 55 ml) was added 0.5 g of activated powdered charcoal (Merck N.F. 18351). After standing I h with intermittent stirring, the mixture was filtered. The charcoal was then washed with IOO ml of phosphate buffer. These adsorption and washing steps freed the malformin fraction almost quantitatively of unconjugated isotopic amino acid. The labeled malformin was extracted from the washed charcoal with 50 ml of acetone. The extract was evaporated to dryness, and the residue was dissolved in I ml of saturated N a H C Q . This latter solution was then extracted five times with 2 ml quantities of ether. The combined ether solutions were in turn extracted twice with I ml portions of 30 % H~S04. The acid extracts were discarded, while the ether solution was dried with 0.5 g of anhydrous Na2SO 4. After removal of the drying agent, the ether was evaporated. The resulting residue of crude malformin was taken up in 2 ml of ethanol, and transferred to a steel planchet. After drying, the radioactivity was measured in a gas flow Geiger counter.

Chromatography o/ labeled mal/ormin In order to better establish the identity of the radioactive material isolated in the above procedure, certain preparations were subjected to descending paper chromatography, using two solvent systems (in separate experiments): (a) Pure water, RF of malformin ~ 0.70-0.80; (b) Isopropanol-NH3-H~O, R F of malformin = 0.87-0.93. A pure sample of malformin A was run as a marker. Its position was revealed b y a chloroplatinate test, as a light region against a pink background g. At the conclusion of the runs, the chromatograms were cut into I-cm wide strips, and the latter were eluted with ethanol onto planchets for drying and counting.

Per/ormic oxidation o/labeled mal/ormin A quantity of crude peptide, representing about 25 ooo counts/min, was mixed with 2 mg of authentic non-isotopic malformin carrier, in 4.5 ml of 98 % formic acid. 0.5 ml of 30 % H20~ was next added, and the mixture allowed to stand for 60 min at room temperature. IO mS of water was subsequently added, and the solution evaporated to dryness in vacuo. The residue was then dried over K O H and P~O5.

Paper electrophoresis o/untreated, and per[ormic-oxidized mal]ormin Further proof of the chemical identity of the labeled peptide was obtained b y subjecting samples of both the - S - S - and - S Q H forms to conventional paper electrophoresis at IOOOV (25 V/cm) for 3 h, using 50 % acetic acid as the "buffer". The position of the oxidized peptide was revealed by its acidic reaction, upon immersion of a guide strip of the dry electropherogram in an alkaline solution of bromophenol blue. A yellow spot was observed against a blue background. Radioactivity was measured as in the case of paper chromatography.

Radioactivity o[ cellular protein The washed mycelium from the malformin assay procedure was homogenized with IO ml of 5 % trichloroacetic acid, followed by heating for 15 min at 9 o°. Then the protein was centrifuged and washed consecutively with (cold) 5 % trichloroacetic

Biochim. Biophys. Acta, 119 (1966) 614-623

MALFORMIN SYNTHESIS IN ASPERGILLUS CELLS

6I 7

acid and ethanol-ether (2:I, v/v). It was finally dissolved in 97 % formic acid, transferred to a planchet, and the latter dried for counting.

Radioactive intracdlular mal[ormin Washed mycelium from the assay procedure was reflnxed for I h in 50 ml of ethyl acetate. After centrifuging, the supernatant solution was concentrated almost to dryness. The concentrate was taken up in IO ml of the usual 0.05 M phosphate buffer, and subjected to the already described procedure for isolation of malformin, beginning with charcoal adsorption.

RESULTS

In seeking optimum conditions for malformin biosynthesis, certain parameters were varied. Fig. I shows that the utilization of isotopic valine was maximal with a quantity of mycelium equivalent to 25 mg dry weight, in a 5-ml volume of buffer solution. With larger quantities of cells, the efficiency of incorporation into malformin decreased. .~

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Biochim. Biophys. Acta, 119 (I966) 614-623

618

M. YOUKIOKA, T. WINNICK

Fig. 2 compares the activity of isolated mycelium in malformin synthesis at three different temperatures. Although Aspergillus is usually cultivated at about room temperature, it is clear that the washed cells in Fig. 2 had highest activity at 37 °, and lowest at 25 °. Fig. 3A describes the growth of Aspergillus cultures at three temperatures. The initial rate was highest at 3 °0 , intermediate at 37 °, and lowest at 25 °. After three days of cultivation, the curves leveled off, with approximately the same final yields of mycelium at 25 ° and 3 o°, and a somewhat lower value for the 37 ° experiment. 8

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Information on the biosynthetic activity of the cells grown at the three temperatures is presented in Fig. 3B. It m a y be seen that the highest activity for malformin synthesis (with the standard 37 ° assay system) was obtained with mycelium derived from 48-h cultivation at 3 o°. Second best results were with 25 ° and 72-h cultures; while the poorest activity was observed with cells from 37 ° cultures (peak at 36 h). A dramatic difference in the relative rates of cyclic peptide and of protein synthesis was observed when mycelium was taken from cultures of different ages. Fig. 4 indicates that the incorporation of [14C]valine into protein was maximal for washed ce~ts taken from 24-48-h cultures. With longer cultivation times, up to 96 h, the protein biosynthetic activity of the ~celts decreased to a moderate extent. By contrast, the malformin synthetic ability of the mycelium was very low until after 48 h. Thereafter the activity increased rapidly, with a peak a t 72 h, and subsequent decline. It will be seen that this curve agrees fairly well vdth that obtained using isotopic leucine in Fig. 3B. Another interesting finding with washed mycelium was that newly-synthesized Biochim. Biophys. Acta, 119 (1966) 614-623

MALFORMIN SYNTHESIS IN ASPERGILLUS CELLS

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malformin passed rapidly into the medium, and did not accumulate to any significant extent within the cells (Fig. 5). The curve for the appearance of labeled extracellular peptide lagged during the first hour of incubation, but rose rapidly thereafter, finally leveling out after about 6 h. The rate curve of ~4C]leucine incorporation into cell protein, given for comparison, differed only in the absence of an fnifial lag. The final levels of radioactivity in the protein were comparable to tllose~ irL t ] ~ [14C~valine experiment of Fig. 4. Fig. 6 provides evidence that t]~e labeled reaction product gor~ted in the usual assay procedure was i n d e ~ ma]/ormm. In chromatography ~ two different solvents, the radioactivity ~ recovered virtually quantitativeiy in a single, welldefined region, with RF identical to that of malformin A. Further support for the chemical identity of the radioactive malformin was obtained by performic acid treatment. Fig. 7 shows that the oxidized peptide migrated toward the anode during paper etectrophoresis, consistent with the expected conversion of the disulfide bridge to sulfonic acid groups. By contrast, the untreated peptide (without electric charge) failed to migrate. Quantitative comparisons of the utilization of different amino acids were not feasible, since racemic compounds were used in some cases, and D- and L-forms in Biochim. Biophys. Acta, 119 (1966) 614-623

620

M. YUOKIOKA, T. WINNICK

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Fig. 6. Identification of radioactive malformin by paper chromatography. Runs were performed on crude peptide fractions (isolated in the customary manner), following standard incubations of washed mycelium with isotopic amino acid. A, [14C]valine-labeled product; solvent: propanolBIHs-H=O. B, [~C]valine (solid lines) and [14CJleucine (broken lines)-labeled products, derived from separate incubation experiments; solvent: H20. The positions of the malformin A markers are indicated by the dark ovals. 2000 E

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others. Other complicating factors were the wide range in specific radioactivity of the amino acids, and the u n k n o w n sizes of the endogenous 12C-pools. Despite these Biochim. Biophys. Acta, II9 (1966) 614-623

MALFORMIN TABLE

SYNTHESIS

IN ASPERGILLUS

621

CELLS

I

INCORPORATION OF VARIOUS AMINO ACIDS INTO MALFORMIN AND CELLULAR PROTEIN The standard

washed

cell system was employed.

Radioactivity (counts/rain)

[14C]Amino acid ( o . 5 , u C )

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complications, the four component amino acids of malformin were found to be utilized to a comparable degree (Table I). Presumably only the L-form of DL-valine and the D-form of DL-Cystine was incorporated. It is not known whether the latter amino acid was reduced to cysteine prior to incorporation. The good utilization of L-leucine implies a rapid inversion of this compound into the D-isomer. Here again it is not clear whether this transformation occurred prior to peptide bond formation. It is noteworthy that D-leucine, unlike the above amino acids, was poorly utilized for protein synthesis, suggesting that the transformation of D- to L-leucine was negligible in Aspergillus cells. The lower half of Table I reveals that the laC of six amino acids which do not occur in malformin A (or B), was not significantly incorporated into the cyclic peptide, although this 14C was very efficiently incorporated into protein (except with tryptophan). These results strongly suggest that the procedure employed measured the biosynthesis of malformin.

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F i g . 8. E f f e c t o f e x c e s s q u a n t i t i e s o f n o n - i s o t o p i c D- o r L - f o r m s o f l e u c i n e o n t h e u t i l i z a t i o n o f r a d i o a c t i v e e n a n t i o m o r p h s o f t h e s a m e a m i n o a c i d (o.5 # C i n e a c h c a s e ) f o r m a l f o r m i n s y n t h e s i s .

Q - O , L-[I'C] and D-[IIC]; (~--C), L-[14C] and L-[12C]; ~k--&, D-[14C] and D-[12C]; /k--A D-[14C] a n d L- [1=C].

Biochim. Biophys. Acta, 1 1 9 ( 1 9 6 6 ) 6 1 4 - 6 2 3

622

M. YUOKIOKA, T. WINNICK

The experiments in Fig. 8 were designed to give clues to the mode of utilization of the D- and L-isomers of leucine for malformin synthesis. It m a y be seen that the uptake o:f D- or L-[14Clamino acid was strongly inhibited by prior dilution with a high proportion of the corresponding non-isotopic isomeric form. The strong inhibitory effi~ct of excess t-IlzClleucine on D-[14C]leucine incorporation m a y very likely reflect the rapid transformation of L- to D-enantiomorph, already cited. However, the partial effect of excess D-[xzCJleucine on the utilization of L-[14Clleucine is presently difficult to explain.

DISCUSSION The selectivity of the isolation and purification procedures employed for malformin, together with the narrow specificity of amino acid utilization, leave little doubt regarding the identity of the labeled product. The remarkably high efficiencies of amino acid incorporation into protein, as well as peptide, suggest t h a t A. niger cells are well suited for further work. The process of malformin synthesis appeared to be quite sensitive to such experimental conditions as the rate of shaking, and the time and temperature of cultivation. This situation is reminiscent of that in cells of B. brevis, in which the formation of gramicidins and tyrocidines is greatly influenced b y the conditions of growth 11. There are obvious and severe limitations inherent in experiments restricted to whole cells. One such difficulty was apparent in connection with the interconversion of D- and L-forms of leucine, and present inability to decide at which level these inversions occurred. Likewise, it could not be determined whether cystine was directly utilized, or formed after the incorporation of two cysteine residues. Another inconclusive experiment involved the addition of actinomycin D, dihydrostreptomycin, chloramphenicol, and puromycin to the washed cell system (data not reported). Even at high concentrations of these antibiotics, there was no significant inhibition of either protein or malformin synthesis over periods up to 12 h. It m a y well be that the antibiotic substances failed to penetrate the Aspergillus cells. The availability of a cell-free system for malformin biosynthesis will permit a variety of basic experiments on the intimate mechanisms of this process. Such studies will be reported in subsequent papers in this series.

ACKNOWLEDGEMENTS This research was supported b y grants from the National Institutes of Health (GM 9335), and the Hawaii Division of the American Cancer Society. The authors are indebted to Dr. R. W. CURTIS, Purdue University, for generously providing much helpful information, including unpublished data. They also thank him for a sample of malformin A and cultures of A. niger. The valuable collaboration of Dr. P. M. RAO in certain of the experiments is appreciated.

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623

REFERENCES

1 A. MEISTER, Biochemistry ot the Amino Acids, 2nd ed., Vol. i, Chapt. 5, A c a d e m i c Press, Mew York, 1965. 2 T. S. EIKHOM AND S. LALAND, Biochim. Biophys. Acta, IOO (1965) 451. 3 M. YUKIOKA, Y. TSUKAMOTO, Y. SAITO, T. TSUJI, S. OTANI AND S. OTANI, Biochem. Bio,phys. Res. Commun., 19 (1965) 2o4. 4 ]3. MACH AND E. L. TATUM, Proc. Natl. Acad. Sci. U.S., 52 (1964) 876. 5 J- B. HALL, J. SEDAT, P. R. ADIGA, I. UEMURA AND T. WINmCK, J. Mol. Biol., 12 (i965) 174. 6 U. SPAERIN, L. O. FROHOLM AND S. F. LALAND, Abstr. Fed. Europ. Biochem. Soc., $6c. Meeting, Vienna, 1965, p. 3. 7 K. ANZAI AND R. W . CURTIS, Phytochemistry, 14 (1965) 263. 8 N. TAKAHASHI AND R. W . CURTIS, Plant Physiol., 36 (1961) 3 o. 9 I. SMITH, Chromatographic and Electrophoretic Techniques, Vol. I, I n t e r s c i e n c e , N e w Y o r k 196o, p. 98. IO M. LEDERER, Chromatographic Reviews, Vol. I, Elsevier, A m s t e r d a m , 1959, p. 46. I I K. OKUDA, G. C. EDWARDS AND T. WINNICK,J. Bacteriol., 85 (1963) 329.

Biochim. Biophys. Acta, i i 9 (1966) 614-623