J. Biochem. 84, 435-441 (1978)

Studies on Gramicidin S Synthetase Purification and Properties of the Light Enzyme Obtained from Some Mutants of Bacillus brevis1 Masayuki KANDA, Kazuko HORI, Toshitsugu KUROTSU, Setsuko MIURA, Akiko NOZOE, and Yoshitaka SAITO 1 Department of Biochemistry, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo 663 Received for publication, February 2, 1978

The phenylalanine-activating and/or -racemizing enzyme, i.e., the light enzyme, of gramicidin S synthetase was purified to a homogenous state by D-phenylalanine-Sepharose 4B chromatography from a wild and some gramicidin S-lacking mutant strains of Bacillus brevis. The light enzyme obtained from a mutant strain E-l could activate phenylalanine but not racemize it, and had no phenylalanine-dependent ATP-[14C]AMP exchange activity, whereas the same enzyme obtained from other mutants and the wild strain had all three activities. Furthermore, the light enzyme of the mutant E-l could form only acid-labile enzyme-bound phenylalanine, while the same fraction of the wild strain carried half of the enzyme-bound phenylalanine as acid-labile adenylate and half as acid-stable thioester. These results suggest that the thiol site of the light enzyme of mutant E-l might be damaged.

Gramicidin S is an antibiotic cyclic decapeptide produced by Bacillus brevis, and its synthetase has been fractionated into light and heavy enzymes by gel-filtration chromatography (1-3). The light enzyme [EC 1.1.11] catalyzes phenylalanine activation and racemization, and the heavy enzyme is an enzyme complex of L-proline-, L-valine-, L-ornithine-, and L-leucine-activating enzymes. In 1 This study was supported in part by a research grant from the Bristol JARA Fund. * To whom inquiries about this paper should be addressed. Abbreviations: DKP, D-phenylalanyl-L-prolyl diketopiperazine; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.

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435

preceding papers from our laboratory (4, 5), it has been reported that twenty mutants of Bacillus brevis which could not synthesize gramicidin S were isolated by N- methyl - iV'-nitro - iV-nitrosoguanidine treatment and classified into five groups according to their characteristic deletion of amino acid-activating enzyme involved in gramicidin S synthesis. Some of these mutants could not even form D-phenylalanyl-L-prolyl diketopiperazine (DKP), though they had apparent phenylalanineand L-proline-activating activities. This paper describes the purification and some properties of the phenylalanine-activating and/or -racemizing enzyme, i.e., the light enzyme, of these gramicidin S-Iacking and DKP (—) mutants as compared to those of the wild strain.

436

M. KANDA, K. HORI, T. KUROTSU, S. M1URA, A. NOZOE, and Y. SA1TO

brought to 45 % saturation of ammonium sulfate, and the mixture was stirred for 30 min and centriMATERIALS AND METHODS fuged. The precipitate was dissolved in a minimum Materials—Uniformly labeled L-[1*C]phenyl- amount of buffer A. The enzyme solution was alanine (480 mCi/mmol), uniformly labeled ["Q- dialyzed overnight against the same buffer and AMP (526 mCi/mmol), and "Pi were purchased applied to a DEAE-cellulose column (1.9x45 cm) from the Radiochemical Centre, Amersham, equilibrated with buffer A. The column was England. Radioactive pyrophosphate was pre- washed with buffer A containing 0.1 M KG and the pared from "Pi by heating at 400°C for 1 h and enzyme was then eluted with a linear gradient of purified by column chromatography on Dowex-1 0.1 to 0.5 M KC1 in buffer A at a flow rate of 50 ml (6). Standard proteins for molecular weight per h. The elution pattern of the wild enzyme is determination were purchased from Boehringer shown in Fig. 1. The enzymes of the wild and Mannheim Gmbh. Other chemicals were usual mutant strains show almost the same pattern. commercial products. The phenylalanine-activating enzyme was comPreparation of Sepharose 4B containing r> pletely separated from an L-proline-, L-valine-, phenylalanine bound through the carboxyl group L-ornithine-, and L-leucine-activating enzyme com(free-amino r>phenylalanine Sepharose 4B) was plex, the heavy enzyme, which was eluted later performed with amino-alkyl Sepharose 4B by than the light enzyme in DEAE-column chrothe method of Pass et al. (7). matography. The activity of the heavy enzyme is Preparation of Sepharose 4B containing r> indicated as L-ornithine-dependent ATP-["PPi] phenylalanine bound through the amino group exchange activity in Fig. 1. (free-carboxyl r>phenylalanine-Sepharose 4B) was The active fractions for D-phenylalanine carried out with succinated amino-alkyl Sepharose activation were pooled and precipitated by addition 4B by the method of Cuatrecasas and Anfinsen of ammonium sulfate to 50% saturation. The (.8). precipitate was dissolved in a minimum amount The amount of amino acid bound to the gel of buffer A, and dialyzed overnight against buffer was estimated by the method of Pass et al. (7). A. The dialyzed enzyme was applied to a freeL-[14C]Phenylalanine was used for this purpose. amino D-phenylalanine-Sepharose 4B column (1.5 x Free-amino phenylalanine-Sepharose contained 45 cm) equilibrated with buffer A and eluted with about 1.2 pimoX of phenylalanine/ml gel, and free- a linear gradient of 0 to 0.25 M KC1 in the same carboxyl phenylalanine-Sepharose contained about 1.5 (tmo\ of phenylalanine/ml gel. These values are comparable to those of Pass et al. (7). Purification of the Enzyme—The mutants and the wild strain of Bacillus brevis described in previous papers (4, 5) were used throughout these experiments, and the cells were cultured and harvested by the methods described in those papers (4, 5). The cells were suspended in 4 volumes of 0.05 M potassium phosphate buffer, pH 7.5, containing 10% glyceiol, 1 mM dithiothreitol, and 50 100 150 1 mM MgCl, (buffer A), and sonically disrupted FRACTION NUMBER at 10 kHz, 100 W, for 4 min at 0°C. All subsequent procedures were carried out at 2°C to Fig. 1. DEAE-cellulose chromatography of gramicidin 3°C. The sonicate was centrifuged at 45,000 x g S synthetase of the wild strain. The procedure is defor 60 min, and solid ammonium sulfate was slowly scribed in the text. Six ml fractions were collected and , added to the supernatant solution with constant aliquots of 0.2 ml were employed for the assay. Absorbance at 280 nm; , concentration of KC1; stirring to give 33 % saturation. The mixture was • , D-phenylalanine-dependeot ATP-"PPi exchange allowed to stand for 30 min, and centrifuged at activity; O, L-ornithine-dependent ATP-**PPi exchange 6,000xg for 30 min. The supernatant was activity. / . Biochem.

THE LIGHT ENZYME OF GRAMICIDIN S SYNTHETASE buffer at a flow rate of 30 ml per h. A characteristic elution pattern of the wild enzyme is shown in Fig. 2. The enzymes of the wild and mutant strains show almost the same pattern. The active fractions were pooled and concentrated by ammonium sulfate precipitation at 50% saturation. The precipitate was dissolved in a minimum amount of 1 mM potassium phosphate buffer, pH 6.5, containing 1 HIM dithiothreitol and 1 mM MgCl,, and dialyzed against the same buffer using a Sartorius collodion bag. The dialyzed enzyme was applied to a free-carboxyl D-phenylalanine-Sepharose 4B column (1.5 x 45 cm) equilibrated with 1 mM potassium phosphate buffer, pH 6.5, containing 10% glycerol, 1 mM dithiothreitol, and 1 HIM MgCl r The enzyme was eluted with a linear gradient of 1 to 50 mM potassium phosphate buffer, pH 6.5, containing 10% glycerol, 1 HIM dithiothreitol, and 1 mM MgClj. The elution pattern of the wild type enzyme is shown in Fig. 3. Again, the enzymes obtained from mutant strains showed almost the same pattern as that of the wild strain. No effect of ATP in the binding of the enzyme to D-phenylalanine-Sepharose 4B was found throughout our r>phenylalanine-Sepharose 4B chromatography. Assay Methods—The assay method for amino acid-dependent ATP-["PPi] exchange was as described previously (9). The determination of phenylalanine racemase was carried out by the method of Yamada and Kurahashi (10). The assay of phenylalanine-dependent ATP-[14C]AMP exchange activity was also performed by the method of Yamada and Kurahashi (70) with some modifications. Separation of adenine nucleotides in the reaction mixture was carried out with a Dowex-1 column (1.0x7 cm, formate form) by the method of Cohn (11). The ATP fraction was concentrated to a small volume with a rotary evaporater, transferred into a planchet and dried, then the radioactivity was measured with a Nuclear Chicago gas-flow counter. The protein concentrations was determined by the method of Kalckar

437

UJ ID

04^- 0 4 5

X O

o CO o 0J 0.2*- 0 2 '

Q.

0

50

100

150

FRACTION NUMBER

Fig. 2. Free-amino D-phenylalanine-Sepharose 4B chromatography of the light enzyme of the wild strain. The procedure is described in the text. Five ml fractions were collected and aliquots of 0.2 ml were employed for the assay. , Absorbance at 280 nm; , concentration of KC1; • , D-phenylalanine-dependent ATP"PPi exchange activity.

UJ


or L-phenylalaninedependent ATP-PC]AMP exchange activities of the light enzymes of the wild strain and mutant strain E-l. The light enzyme of the wild strain had a high exchange activity, while that of the mutant strain E-l did not have any significant activity. The activity of D-phenylalanine-dependent exchange was about twice that of L-phenylalanine-dependent exchange in the case of the wild strain. This result corresponded with the finding that the ratio of L-phenylalanine/o-phenylalanine is about threesevenths at equilibrium of the racemase reaction (10). Table UJ shows that the light fraction of the wild strain contains half of the enzyme-bound

440

M. KANDA, K. HORI, T. KUROTSU, S. MIURA, A. NOZOE, and Y. SATTO

TABLE in. Formation of protein-bound phenylalanine by the light enzyme. The assay was carried out as described in the text. 10 fi% of homogenous enzyme was used for the assay. Enzyme source

L-P*CJPhenylalanine bound to enzyme as Adenylate (nmol)

Thioester (nmoO

Wild strain

0.101

0.107

Mutant E-l

0.079

0.002

["C]phenylalanine as acid-labile adenylate and half as acid-stable thioester, whereas the same fraction from the mutant strain E-l contains only acidlabile adenylate. The light enzyme of racemization ( + ) mutants gave nearly the same results as that of the wild strain, and the enzymes of other racemization (—) mutants showed results similar to those for the mutant E-l, although these results are not presented in Tables II and HI. DISCUSSION Phenylalanine-activating and/or -racemizing enzyme, i.e., the light enzyme, of gramicidin S synthetase has been purified to near homogeneity in other laboratories (10, 16). However, our purification procedure presented in this paper is simple and quick, and the specific activity and yield of our homogenous enzyme are somewhat higher than those of other authors (10,16). Amino acid-Sepharose chromatography was first applied to purify gramicidin S synthetase by Pass et al. (7). They found that the light enzyme of gramicidin S synthetase showed affinity for free-amino D-phenylalanine-liganded Sepharose, and the heavy enzyme of gramicidin S synthetase showed affinity for Sepharose carrying L-proline, L-valine, Lornithine, or L-leucine bound through the carboxyl group. They also reported that the presence of ATP is required for binding of the enzyme to the column to occur. Free-amino and free-carboxyl D-phenylalanine-Sepharose 4B were used successively in our phenylalanine-Sepharose chromatography. Free-carboxyl D-phenylalanine-Sepharose 4B chromatography was effective in removing traces of contaminating protein. Contrary to the results reported by Pass et al. (7), no effect of ATP

in the binding of enzyme to free-amino and freecarboxyl D-phenylalanine-Sepharose was found in our experiments. Thus, it is considered that ion exchange by D-phenylalanine bound to the Sepharose column may play an important role rather than affinity of the enzyme for the matrix. Twenty mutant strains of Bacillus brevis which could not form gramicidin S were isolated in our laboratory, and fourteen of them contained the light enzyme. In this work, it was found that the light enzymes of four DKP (—) mutants could activate phenylalanine but not racemize it. Furthermore, the light enzyme of phenylalanine racemization (—) mutants could not catalyze phenylalanine-dependent ATP-["C]AMP exchange and did not form the phenylalanine thioester enzyme complex. It has been postulated that phenylalanine, ATP and the light enzyme form an aminoacyl adenylate enzyme complex with the liberation of inorganic pyrophosphate. The adenylatebound phenylalanine is then transferred to a sulfhydryl group of the enzyme to form a thioester with liberation of AMP, and D-phenylalanine formed is transferred to the heavy enzyme of gramicidin S synthetase (14). It has also been suggested that racemization of phenylalanine takes place on the phenylalanyl thioester enzyme complex (17). It was unsuccessful to separate the phenylalanine-activating activity from the racemizing activity of purified light enzyme (10). In our experiments, it was found that the molecular weight of the light enzyme of the wild strain was identical with that of the enzymes of racemization (—) mutants, suggesting that the activating and racemizing activities are catalyzed by a single enzyme. In a previous paper (5), it was shown that the enzymes of mutant strains E-l, BI-4, C-3, and E-2 could not form DKP, although they had high activity of phenylalanine- and proline-activating enzymes, suggesting that the thiol sites of the light enzymes of these mutants might be damaged so that D-phenylalanine could not be formed from the L-isomer, or could not be transferred to the heavy enzyme. The results in the present paper give strong support to this possibility, since the light enzyme of racemization (—) mutants could not catalyze either the racemization of phenylalanine or phenylalanine-dependent ATP-[14C]AMP exchange, and /. Biochem.

THE LIGHT ENZYME OF GRAMICIDIN S SYNTHETASE

did not form the phenylalanine thioester complex. The estimation of free sulfhydryl groups and amino acid analysis are now in progress with our purified light enzymes obtained from these wild and mutant strains of Bacillus brevis. REFERENCES 1. Tomino, S., Yatnada, M., Itoh, H., & Kurahashi, K. (1967) Biochemistry 6, 2552-2560 2. Saito, Y., Otani, S., Jr., & Otani, S. (1970) Advances in Enzymology (Nord, F.F., ed.) Vol. 33, pp. 337380, Intcrscience Publishers, New York 3. Lipmann, F., Gevers, W., Kleinkauf, H., & Roskoski, R., Jr. (1971) Advances in Enzymology (Meister, A., ed.) Vol. 35, pp. 1-34, Interscience Publishers, New York 4. Iwaki, M., Shimura, K., Kanda, M., Kaji, E., & Saito, Y. (1972) Biochem. Biophys. Res. Commun. 48,113-118 5. Shimura, K., Iwaki, M., Kanda, M., Hori, K., Kaji, E., Hasegawa, S., & Saito, Y. (1974) Biochim. Biophys. Ada 338, 577-587

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6. Berg, P. (1958) / . Biol. Chem. 233, 601-607 7. Pass, L., Zimmer, T-L., & Laland, S.G. (1973) Eur. J. Biochem. 40, 43-48 8. Cuatrecasas, P. & Anfinsen, C.B. (1971) Methods in Enzymology Vol. 22, pp. 345-378, Academic Press, New York 9. Otani, S., Yamanoi, T., & Saito, Y. (1970) Biochim. Biophys. Ada 208, 496-508 10. Yamada, M. & Kurahashi, K. (1969) /. Biochem. 66, 529-540 11. Cohn, W.E. (1957) Methods in Enzymology Vol. 3, pp. 724-743, Academic Press, New York 12. Kalckar, H.M. (1947) /. Biol. Chem. 167, 461-486 13. Omstein, L. (1964) Ann. New York Acad. Sci. 121, Art. 2, 321-349 14. Weber, K. & Osborn, M. (1969) /. Biol. Chem. 244, 4406-4412 15. Roskoski, R., Jr., Gevers, W., Kleinkauf, H., & Lipmann, F. (1970) Biochemistry 9, 4839-4845 16. Vater, J. & Kleinkauf, H. (1976) Biochim. Biophys. Ada 429, 1062-1072 17. Takahashi, H., Sato, E., & Kurahashi, K. (1971) / . Biochem. 69, 973-976