Substrate amino acid-mediated stabilization of gramicidin S synthetase activity against inactivation in vivo

Substrate amino acid-mediated stabilization of gramicidin S synthetase activity against inactivation in vivo Spyridon N. Agathos* and Arnold L. Demain Fermentation Microbiolog'y Laboratory, Department of Applied B&logical Sciences, Massachussets Institute o f Technology, Cambridge, MA 02139, USA (Received 4 October 1985: revised 4 March 1986) We have examined the influence o f the substrates o f gramicidin S synthetase upon stability o f its in vivo activity. A mixture o f L-phenylalanine, L-proline, L-leucine, L-ornithine and L-valine at 6 mM each added to aerated suspensions o f Bacillus brevis cells substantially protected the synthetase activity against oxygen-dependent inactivation. After six hours o f incubation, more than 70% o f the initial activity remained in the amino acid-supplemented vessels, whereas the unsupplemented control lost syn thetase activity with a half-life o f approximately one hour. Omission o f L-leucine or L-ore ithine resulted in diminished protection o f the synthetase activity, whereas these two amino acids provided, synergistically, almost as good a stabilization as did the mixture o f the five amino acids. The amino acid-mediated stabilization was independent o f growth and de novo protein syn thesis. Keywords: Enzyme inactivation #t vivo; gramicidin S synthetase; Bacilhts brevis; substrate-mediated stabilization; amino acid-dependent protection

Introduction Enzyme levels in microorganisms are known to be controlled through regulation of the rate of their synthesis (inductionrepression), whereas enzyme activity is controlled by noncovalent binding of various ligands (activation-inhibition). A reasonable understanding of these control modes is the key to maximizing production of a desired primary or secondary metabolite or a commercially important enzyme. ~ However, an additional type of enzyme regulation is operative in both prokaryotic and eukaryotic microbes, i.e. the control of enzymatic activity by selective inactivation in vivo. This disappearance of particular enzymes is widespread among microorganisms and involves several distinct mechanisms, in many cases inadequately understood or even totally unknown.: Particularly transient in their presence in vivo are the synthetases catalysing the formation of secondary metabolites, such as antibiotics) Despite its obvious importance, in vivo enzyme inactivation in secondary metabolism has received only scant attention, hence the mechanism(s) responsible for the disappearance of antibiotic synthetases remains obscure. Increased understanding of the process would make it possible to achieve prolonged periods of synthesis of a desired metabolite. The only reports specifically addressing the bz vivo stability of antibiotic synthetase activities have come front studies in this

*Present address: Department of Chemical and Biochemical Engineering, Rutgers University, Busch Campus, P.O. Box 909, Piscataway, NJ 08854, USA

0141-0229/86/080465-04 $03.00 © 1986 Butterworth & Co (Publishers) Ltd

laboratory on gramicidin S (GS) synthetase 4-6 and l'rom studies by Gaucher and colleagues 7-8 on patulin synthesis. The present study is a continuation of our investigation on the in vivo inactivation of GS synthetase activity, the enzyme complex catalysing the production of the cyclic peptide antibiotic GS in Bacillus brevis. 4 The inactivation is an oxygen-dependent process 4 which we have recently shown to be modulated by the intracellular redox state of the cells. 9 Organic thiols added to partially anaerobic cell suspensions are capable of reversing the loss of synthetase activity in short-term experiments or delaying it in longterm experiments. Exogenous utilizable carbon sources retard the rate of inactivation in cells of B. brevis incubated for short periods under air. 9 We report here the stabilization of GS synthetase activity through addition of the five amino acids substrates to aerated suspensions of B. brevis. The overall enzymatic reaction catalysed by the synthetase (a complex consisting of a heavy fraction of 280 000 daltons and a light fraction of 100000 daltons) is as follows: 2 2 2 2 2

L-Phenylalanine L-Proline GS synthetase L-Valine +10 ATP GS+ 10AMP+ 10PP i L-Ornithine Mg2 ÷ L-Leucine

Materials and methods Strains, media and g r o w t h c o n d i t i o n s [3. brevis ATCC 9999 was used for the synthesis of GS and its synthetase complex. The organism was maintained as a spore suspension at --20°C. The media and conditions

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Papers for spore preparation, inoculum preparation and fermentations leading to the production of B. brevis cells that conrain GS synthetase activity have been reported previously. 4

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Procedures Jbr study o f inactivation in whole cells The methodology for studying the kinetics of synthetase inactivation has been previously described. 9 Briefly, cell paste of B. brevis that had been stored fiozen at 2 0 ° C (which retains GS synthesizing capacity for at least 12 months) was mixed with buffer A (10 mM triethanolamine chlo,ide. 10 mM MgCI 2, 0.75 mM EDTA, pH 7.6) in tire proportion of 1 g wet cell weight into 3 ml buffer. Typically 12 g wet cells were shaken under air in 36 ml buffer A in a 500 ml baffled Erlenmeyer flask. Aliquots of 4 ml were harvested at different times, treated with lysozyme (2 mg ml -~ ) to produce cell lysates which were subsequently centrifuged to yield supernatant fluids containing the synthetase. These were assayed for GS synthetase activity.

Assay oJ" GS synthetase activity" The method used for determination of synthetase activity has been described previously. 9 It is based on the incorporation of a radiolabelled precursor amino acid into GS, which is isolated as trichloroacetic acid-insoluble material. Protein was assayed by the biuret method of Gomall et al. m The standard reaction mixture for the synthetase assay (labelled assay mix, LAM) (total reaction volume 0.5 ml per assay tube) contained 6.0 mM each of the amino acids L-phenylalanme, L-proline, L-valine and L-leucme (or L-ornithine), 2.0 mM each of t,-ornithine (or L-leucine) plus 0.5 tzCi nil-' of L-[ '4C] ornithine (or L-['4C] leucine), 15ram ATP, 10ram MgCI,, 50ram triethanolamine (ptl 7.6) and 10 mM dithiothreitol (1)TT). Since the cellfree extracts from cells aerated with exogenous amino acids were prepared without removal of the added acids, the aliquots from control vessels (i.e. cells aerated without amino acids) were supplemented with identical amino acid concent,-ations prevailing in the experimental assay tubes.

Results

l(ffect o f the five amino acid substrates on the stability o f GS syn thetase activity It has been recognized that substrates, cofactors and other natural ligands may increase enzyme stability, xa-12 In our case, it was important to determine whether the five amino acid substrates of GS synthetase have any effect on in vim inactivation under air. We thus examined the effect of addition of a mixture of l.-phenylalanine, L-proline, L-valine, l.-ornithine and L-leucine to aerated suspensions of B. brevis frozen-thawed cells. A concentration of 6 mM for each amino acid was chosen to ensure saturation of the enzyme complex, in view of reported Km values for these substrates of at least one order of magnitude lower. 13-~4 ha a series of experiments, it was discovered that stabilization of the synthetase activity occurred (Figure 1). In some cases, not only stabilization, but an apparent activation (i.e. a measurable increase in activity compared to initial activity at zero time) was observed during the incubation. Recent experiments (J. Tasker and S.N. Agathos, unpublished results) indicate that tiffs amino acid-dependent activation occurs in cells in which synthetase had not reached its peak specific activity in the course of the primary cultivation from which the cells originate.

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Hours Figure 1 Effect of five amino acids on synthetasestability under aeration in the presence and absence of chloramphenicol (CAP). L-Phe, L-Pro, L-Val, L-Orn, and L-Leu were added at 6 mM each. Time samples (4 ml) from one control (unsupplemented) vessel were assayed in tubes supplemented with the same concentrations of the amino acids present in the assay tubes from samples originating from the experimental vessels. Agitation was done in air at 300 rev rain -z, 37°C. /-', 5 amino acids (6 mM); A, 5 amino acids ( 6 m M ) + CAP (500/~g ml- ~ ); o, CAP (500 #g ml- ~ ); % no addition

The experiment on amino acid mediated stabilization has been carried out many times. Although the time courses of inactivation and stabilization differ somewhat from experiment to experiment, in every case there was inactivation in the absence of amino acids and stabilization when the five amino acids were added. In most cases, there was no activity loss in the presence of the substrates throughout the duration of the experiments; even in the "worst' case. more than 70% of the initial activity was still present in the cells after 6 h aerobic incubation. This amino acid-mediated stabilization is thus much more pronounced than that previously obsel~,ed with utilizable carbon sources. 9

h~dependence o f amino acid-mediated stabilization o f the synthetase activity in v i v o from cell growth and protein sy n thesis We next investigated whether the stabilization required or was even accompanied by an increase in cell mass or protein. Absorbance measurements throughout the duration of the aerobic incubation in the presence and absence of added amino acids indicated no increase in cell mass. Indeed it was repeatedly found that the final absorbance values were at least 3ff2~ lower than at zero time, indicating the occurrence of lysis during the agitation period (rest, Its not shown). The qt,estion of whether de novo protein synthesis was occurring during incubations with the five amino acids was studied with the use of the protein synthesis inhibitor chlorampbenicol (CAP). As seen m Figure 1, 500 #g ml -~ CAP did not abolish the protective effect of the amino acids on the synthetase activity. It should be noted that the CAP was used in excess since the mi,timal inhibitory concentration of CAP for B. brevis is lower than 100/~g m1-1 (data not shown). We also followed the incorporation of 14C-labelled [.-leucine into trichloroacetic acid (TCA)-precipitable material. No incorporation was observed in the presence or absence of CAP during the first 6 h. Incorporation was only observed after at least 12 h. From all the above lines of evidence, it is concluded that the five constituent amino acids of (iS are able to stabilize lit vivo the synthetase activity against O2-dependent inactivation in tire absence of growth or protein synthesis. r h e protection of the synthetasc activity by the five amino acid substrates was found to be concentration-

Stabilization of gramicidin S synthetaseactivity: S.N. Agathos and A. L. Demain Ioo

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Figure 2 Effect of mixture of L-leucine and L-ornithine on GS synthetase stability in vivo under aeration, o, no addition; ~, L-Leu, 6 mM; ~, 5 amino acids, 6 mM each; v, L-Leu, 6 mM + L-Orn, 6mM

dependent. Whereas concentrations of 0.5 and I mM provided only small retardations of tile inactivation, good protection was achieved when the five substrates were added at concentrations of 3 to 6 mM each.

Relative i m p o r t a n c e o f different a m i n o acids The next question was whether all five amino acids are necessary for the observed stabilization. A series of tlasks was used each containing different mixtures of four amino acid substrates, along with two control flasks, one supplemented with all five amino acids and one with no amino acids. The results (data not shown) indicated that omission of either L-phenylalanine. e-p,-oline or L-valine has no effect on stabilization. Interestingly. these are tile first three amino acids incorporated in the process of GS biosynthesis. On the other hand, omission of t.-leucine or L-ornithine resulted in a decrease in stabilization. Since I.-leucme and L-ornithine appear to have special significance in the stabilization effect, we tested whether leucine or ornitbine by itself could stabilize tile enzyme. Although ornithine was inactive, leucine at 6 mM showed a positive retardation effect but not as great as shown by tile five amino acids. L-Leucine was tested also at 30 lnM i.e. equivalent to the overall concentration of the five added amino acids. However, enzyme inactivation in this case did not differ from that observed with 6 mM L-leucine. In view of these findings we tested whether leucinc and ornithine, added together, could effect greater stabilization of the enzyme activity. As seen in l~Tgure 2, the combination of t,-leucine and L-ornithine (at 6 mM concentration each) was much better than leucine alone and was able to retard the inactivation quite significantly. In this experiment, the estimated half-life of the enzyme activity in the control vessel, i.e. 70 rain, was marginally increased by L-leucine to 90 rain. An improvement to 5 h occurred in the presence of l.-leucine plus L-ornithine, whereas the extrapolated h a l f life with all five amino acids was about 9 h. These results suggest that L-leucine and L-ornithme have special significance as stabilizers of the synthetase activity.

Discussion The five amino acid substrates of GS synthetase were shown to protect in vivo the activity of the enzyme complex against 0 2 . On the basis of known facts about the enzymo-

logy of GS biosynthesis, our data on substrate-mediated stabilization can be used to explain the stabilization effect. The biosynthesis of the decapeptide antibiotic requires the close association of two polyenzymes, heavy GS synthetase and light GS synthetase. These muhifunctional proteins are not resolvable into subunits by conventional denaturing agents such as sodium dodecy[ sulphate. ~s-~7 Studies on limited proteolysis of the heavy enzyme as well as those on defective enzymes from mutants have revealed that the heavy enzyme is made up of a single polypeptide chain consisting of a series of 'subunits' or. more aptly, 'functional domains', that are linked sequentially to each other through covalent bonds. ~8 Each of these functional domains is characterized by a primary amino acid-activating function independent of the total peptide-forming ability of the complex. ~7 The evidence isconsistent with a sequence of the domains (activating units) identical with the amino acid sequence in GS. ~v-'8 At least one sulphydryl group in each activating site is essential /'or thioester formation with the corresponding amino acid. "lhese functional groups are prime candidates for targets of Oz-dependent inactivation. Our own work 9 has provided evidence on SH group involvement. At high substrate and ATP concentrations (at least 10 times the K m values), there has been reported some increase in in vitro stability of the synthetase activity ~9 probably due to protection of SH groups on the active sites by thioesterified amino acid substrates. 2°-2a The protection of SH groups through corresponding amino acid thioester formation may explain why enzyme isolation and purification procedures commonly reported, which make no effort to exclude air, still yield active GS synthetases. This type of mechanism, i.e. essentially a saturation of the activating domains of the heavy enzyme polypeptide with the four amino acids and of the single functional domain of the light synthetase with L-phenylalaninc. might explain the sizeable protection in vivo against 02 reported in this connnunication. Moreover the presence of all five amino acid substrates would appear to ensure the best stereochemical fit into the respective cavities of the multienzyme template and guarantee not only catalytic but also structural integrity of the synthetases. Our data point to some special status of leucine and ornithine insofar as their interaction with the multifunctional enzyme is concerned. This special status of [eucine and omithine in their protective effect cannot be explained by differences in binding constants between these and tile other three substrate amino acids.'3-~4 From the work of other investigators 18"22-29 it would appear that the ornithine- and leucine-activating units of the heavy multienzyme are of particular importance for tile structural integrity (ornithine) and tile catalytic efficiency (leucine) of the synthetase complex. Thus, omission of either of the two amino acids might lead to a certain vulnerability of the synthetase activity to O2-dependent inactivation. Moreover, replacement of the five amino acid substrates by ornithine plus leucine results in almost the same degree of stabilization, presumably due not only to the special attributes ascribed above to their respective active sites, but also possibly because of some additive conformational interaction stabilizing the whole polyenzyme complex w h e n these two active units are both occupied. Although synergistic effects of two ligands upon the stability of an enzyme activity are generally not well understood, they are thought to involve an overall conformational change (stereochemical reorientation) of the enzyme in a manner

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Papers

that makes it less vulnerable to the inactivating principle. For example, AMP and fructose 2,6-bisphosphate were reported to have a synergistic effect against inactivation of fructose 1,6-bisphosphatase by trypsin, 3° a phenomenon attributed to either a mutual enhancement of the affinity of the enzyme by each of the two compounds for the other or to a joint inducement of a unique conformational state that is highly stable to tryptic digestion.

Conclusions The main significance of our research is as follows: (a) stabilization by substrate amino acids occurs in a 'natural' system, well suited for antibiotic production (as opposed to anaerobiosis) s ; (b) amino acid stabilization data point to the thiol groups on the amino acid activating centres as the targets of" the O2-induced inactivation in vivo and also independently agree with established facts from the enzymology of GS synthetase active sites; and (c) amino acid protection can be potentially useful in purification of GS synthetase and related peptide antibiotic synthetases for synthetic applications.

Acknowledgements We express our appreciation to AiQi Fang for expert technical assistance. We thank A.M. Klibanov and G.M. Whitesides for useful discussions. This research was supported, in part, by a NATO Science Fellowship to S.N. Agathos through the Ministry of National Economy of Greece.

References 1 2 3

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9 Demain, A. L. and Agathos, S. N. Can. J. Microbiol. in press 10 Gornall, A.G., BardawiU, C. and David, M. J. Biol. Chem. 1949, 177,751 11 Grisolia, S. Physiol. Rev. 1964, 44,657 12 Wiseman, A. in Topics in Enzyme and Fermentation Biotechnology (Wiseman, A., ed.) Hlis llorwood, Chichester, 1978, vol. 2, pp. 280 -303 13 Zimmer, T.-L. and Laland, S.G. Methods l'.nzymol. 1975, 43, 567 14 Stramondo, J.G. PhD Thesis, M.I.F., Cambridge, Massachusetts, 1979 15 Koischwitz, H. and Kleinkauf, H. Biochim. Biophys. Acta 1976, 429, 1052 16 Christianscn, C., Aarstad, K., Zimmer, T.-L. and Laland, S.(;. FEBS Lett. 1977, 81,121 17 Kleinkauf, H. and Koischwitz, H. in Cell Compartmentation and Metabolic Channeling (Nover, L., Lynen, P. and Mothes, K., eds) l-lsevier/North Holland Biomedical Press, Amsterdam, 1980, pp. 147-158 18 Kleinkauf H. and Koischwitz, H. in Multifunctional Proteb~s (Bisswanger, H. and Schmincke-Ott, E., eds) John Wiley and Sons, New York, 1980, pp. 217-233 19 Kleinkauf, H. and von DOhren, H. in Adr'ances in Biotechnology (Vezina, C. and Singh, K., eds) Pergamon Press, Toronto, 1981,vol. 3, pp. 83 88 20 Otani, S., Yamanoi, Y. and Saito, Y. J. Biochem. (7okyoJ 1969, 66,445 21 Kristensen, T., Gilhuus-Moe, C.C., Zimmer, T.-L. and Laland, S.G. Eur. J. Biochem. 1973, 34,548 22 Vater, J. and Kleinkauf, H. Acta Microbiol. Acad. Sei. flung. 1975, 22,419 23 Kleinkauf, H., Koischwitz, H., Vater, J., Zocher, R., Keller, U. Mahmutoglu, I., Bauer, K., Altmann, J., Kittelberger, R., Marahiel, M. and Salnikow, J. in Reg~dation o f Seeondao' Product and Plant Hormone Metabolism (Luckner, M. and Schreiber, K., eds) Pergamon Press, Oxford, 1979, pp. 37-47 24 Kittelberger, R., Koischwitz H. and Kleinkauf tl. presented at Xlth International Congress of Biochemistry, Toronto, Canada, July 8-13, 1979 Abstract No. 04 5-S108 25 Altmann, M., von Dohren, H., El-Samaraie, A., Pore, M., Kittelberger, R. and Kleinkauf, I1. in Peptide Antibiotics Biosynthesis and l.'uncn'ons (Kleinkauf, H. and yon Dcihren, I1., cds) Walter de Gruyter, Berlin, 1982, pp. 243--252 26 ttori, K., Kurotsu, T., Kanda, M., Miura, S., Nozoe, A. and Saito, Y.J. Biochem. (7bkyo), 1978, 84,425 27 Pass, [.., Zimmer, T.-L. and Laland, S.G. Eur. J. Biochem. 1973,40,43 28 Kambe, M., Imae, Y. and Kurahashi, K. J. Bioehem. (Tok3'o), 1974, 75,481 29 Laland, S.G., Aarstad, K. and Zimmer, T.-L. in Peptide Antibioties - Biosynthesis and Functions (Kleinkauf, H. and von Dohren, H., eds), Walter dc Gruyter, Berlin, 1982, pp. 185-194 30 Hart, R.F.. Han, G.Y., Hayes, R.L., Moore, C'.L. and Johnson, J. l:'xperientia 1983, 39, 1305