Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki

Agric. Biol. Chem., 42 (1), 95 `100, 1978 Pathway and Regulation of Lysine Biosynthesis in Brevibacterium Iactofermentum õ Osamu TOSAKA and Koichi TAKINAMI Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki Received August 16, 1977 The pathway and regulation of lysine biosynthesis in Brevibacterium lactofermentum were investigated. The biosynthetic pathway of L-lysine in Brevibacterium was the same as that in Escherichia coli, since biosynthetic enzymes, which were aspartokinase [EC 2.7.2.41, dihydrodipicolinate (DDP)* synthetase, DDP reductase and N-acetyl-ƒÃ-keto-ƒ -aminopimelate (AKAP) synthetase, could be detected in B. lactofermentum. AKAP synthetase was measured with ' -piperideine-2,6-dicarboxylate, which was prepared from L-diaminopimelate by L-amino acid oxidase, and either acetyl-coa or succinyl-coa could be served as an acyl donor for this reaction. Effect of amino acids on these enzymes was estimated. Aspartokinase was inhibited about 41, 45 and 82% by single or simultaneous addition of L-threonine and L-lysine at l mm, respectively, whereas the addition of other amino acids did not cause significant inhibitory effect. In contrast with DDP synthetase from E. coli, B. lactofermentum enzyme was not affected by L-lysine. DDP reductase was not inhibited by L-lysine, but about 56 and 47% by L-cysteine and L-alanine at 10 mm, respectively. AKAP synthetase was not affected significantly by L-lysine at 10 mm. From the above results, the regulation mechanism of lysine biosynthesis in B. lactofer mentum was discussed. The pathway of lysine biosynthesis in microorganisms was first reported by Gilvarg et al1) using a coliform bacteria representative of the principal genera of the Enterobacteriaciae. It has been well known that there are two different pathways of lysine biosynthesis. One is the diaminopimelate pathway in bacteria, the other is the ƒ -aminoadipate pathway in fungi and yeasts. Shiio et al. reported pre viously that lysine biosynthetic enzymes such as aspartokinase, aspartate-ƒà-semialdehyde de hydrogenase, DDP synthetase and DDP reductase were presented in B. flavum.2,3) Therefore, it was suggested that the pathway of lysine biosynthesis in B. flavum would be the same as that in E. coli. On the other hand, in any microorganisms, there were no reports to examine the enzyme which forms N-acetyl-ƒÃketo-ƒ -aminopimelate from ' -piperideine-2,6- dicarboxylate. This paper deals with the properties of lysine biosynthetic enzymes in B. lactofermentum; they are aspartokinase, DDP synthetase, DDP reductase and AKAP synthetase. r Biosynthesis of L-Lysine and L-Threonine in Brevibacterium. Part I. * The following abbreviations were used in this report: DDP, dihydrodipicolinate; AKAP, N-acetyl-ƒÃketo-ƒ -aminopimelate; ASA, aspartate-ƒà-semialdehyde; DAP, ƒ,ƒã-diaminopimelate; Asp, aspartate; Ala, alanine; Arg, arginine; Glu, glutamate; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Met, me thionine; Pro, proline; Ser, serine; Val, valine; Lys, lysine; Thr, threonine; ATP, adenosine 5'-triphosphate, ADP, adenosine 5'-diphosphate; AMP, adenosine 5'-monophosphoric acid, MATERIALS AND METHODS Microorganisms and culture conditions. Brevibac terium lactofermentum AJ-1511 and the homoserine requiring mutant (A7 `3270) were inoculated in 20 ml medium of the following composition: glucose, 100 g; ammonium sulfate, 45 g; KH2P04, 1 g; MgSO4 E7H20, 0.4g; FeSO4 E7H2O, 10mg; MnSO4 E4 `6H2O, 8.16mg; thiamine HCl, 100 µg; d-biotin, 50 µg per liter; and ph 8.0. CaCO3 was added to make 5 % to prevent

96 0. TOSAKA and K. TAKINAMI the exessive ph shift of the medium from neutral. In the case of strain AJ-3270, 200 mg/liter of L -threonine and 200 mg/liter of L-methionine were added to above medium. These bacteria were grown in 500 ml flask on a shaker at 30 C. Enzyme preparation. After cultivation for 24 hr, the grown cells were harvested and washed twice with 0.2 % of KCl. Then the washed cells were suspended in 0.05 M K-phosphate buffer, ph 7,0 and sonifically disrupted in ice bath. After the centrifugation at 20,000 x g for 30 min at 0 C, the supernatant was mixed at 0 C with 5 volumes of saturated ammonium sulfate solution. After stirring for 30 min at 0 C, the precipitates were collected by the centrifugation at 20,000 x g and dis solved in the same buffer. The solution (15 to 20 mg protein per ml) from B. lactofermentum AJ-1511 was used as the crude enzyme preparation unless otherwise cited. Enzyme assay. (1) Aspartokinase activity was determined by measuring the aspartohydroxamate ac cording to the method of Black and Wright.4) The assay mixture contained following components in a total volume of 2 ml: L-aspartate, 50 µmoles; ATP, 30 µmoles; MgS04 E7H20, 20 µmoles; hydroxylamine, 500 µmoles; ammonium sulfate, 400 µmoles; Tris H2SO4 buffer (ph 7.5), 100 µmoles and enzyme. After incubation at 37 C for one hr, the reaction was stopped by adding 3 ml of FeC13 reagent. After centrifugation, the absorbancy was measured at 540 nm. A blank reaction mixture without L-aspartate served as a control. (2) DDP synthetase was estimated by the same methods of Yugari and Gilvarg.5,6) The reaction mix ture contained 200 µmoles Tris-HCl buffer (ph 8.0), 1 ƒêmole sodium pyruvate, 1 µmole ASA and the crude units of L-amino acid oxidase and 60 units of catalase in a total volume of 1 ml. After incubation for 90 min at 30 C, 0.5 ƒêmole of succinyl-coa (or acetyl-coa), 35 moles of KCl and enzyme were added. Incu bation was carried out at 37 C for 10 min. After the reaction was stopped by the addition of 25 % TCA (0.25 ml), the reaction mixture was centrifuged for 5 min at 10,000 x g. The supernatant was mixed with 0.1 ml of 1 mm DTNB. The optical density was meas ured at 412 nm. Protein estimation. Protein was estimated by the method of Lowry et a1.7) RESULTS Aspartokinase In B. flavum, it has been reported that the aspartokinase was sensitive to the feedback inhibition in the simultaneous presence of L- threonine and L-lysine, and this enzyme was the main regulatory site in the lysine biosyn thesis." In order to examine the properties of the aspartokinase of B. lactofermentum, the enzyme was partially purified. Requirements for aspartokinase are shown in Table I. This enzyme required ATP, aspartate and Mg2+ for its reaction, and ammonium sulfate did not affect the activity significantly. TABLE I. REQUIREMENTS FOR ASPARTOKINASE ACTIVITY enzyme in a total volume of 1 ml. After incubation for 10 min at 30 C, the reaction was stopped by the addition of 1 ml of 1 N HCl. Then o-aminobenzaldehyde (0.2%) was added to the reaction mixture. After standing for 50 min at room temperature, the mixture was centrifuged at 3000 rpm for 10 min. The increase of the optical density at 540 nm was measured on Hitachi-recording spectrophotometer Eps-3. (3) DDP reductase was measured by the coupled method. The reaction mixture contained 100 µmoles of Tris-HCl buffer (ph 7.5), 10 µmoles of DL-ASA, 5 µmoles of pyruvate and enzyme in a total volume of 0.9 ml. After standing for 5 min at room temperature, 0.1 µmole of NADPH was added to give a final volume of 1.0 ml, and then the decrease of optical density at 340 nm was determined. (4) AKAP synthetase was measured by the coupled method. The reaction mixture contained 80 moles of Tris-HCl buffer (ph 7.5), 0.4 ƒêmole of DAP, 10 a Complete system was as follows : Tris -H2SO4 buffer (ph 7.5), 100 µmoles; L-aspartate, 50 ƒê moles; ATP, 30 µmoles; MgSO4 E7H2O, 20 µmoles; hydroxylamine, 500 µmoles; (NH4)2S04, 400 ƒêmoles and enzyme in a total volume of 2 ml. Table II shows the effect of amino acids on aspartokinase under the standard assay con ditions except that the concentration of aspartate was 10,ƒÊmoles. One mm of lysine and threonine inhibited aspartokinase about 45 and 41 %, respectively, as compared with no addition. On the other hand, the simultane-

Biosynthesis of L-Lysine and L-Threonine in Brevibacterium 97 TABLE II. EFFECT OF AMINO ACIDS ON ASPARTOKINASE Assay methods were the same as those of Table I except that the concentration of L-aspartate was 10 moles and the amino acids were added as indi cated. Amino acids were all in L-form. biosynthesis, is strongly inhibited by lysine," while those of B. flavum,8) Bacillus subtilis,9) Corynebacterium glutamicum10) or Strepto coccus faecalis11) are not. Furthermore, the formation of this enzyme is repressed by lysine in S. faecalis and Staphylococcus aureus, but not in Bacillus subtilis and B. flavum. Thus, it is of interest to elucidate the regulation of DDP synthetase in B. lactofermentum. The condensation product was identified as DDP by comparing its o-aminobenzaldehyde in neutral and acidic ph regions with the results of Yugari and Gilvarg.5) The specificity of ƒ -keto acid for DDP synthetase was shown in Table IV, which shows that this enzyme requires pyruvate specifically. As shown in Fig. 1, the optimum ph for DDP synthetase was 8.2 in Tris-HCl buffer when measured by the coupled assay method. Moreover, the effect of lysine on the activity of DDP synthetase was investigated at various ph. Effect of lysine was not observed and not so affected by varying the ph of reaction mixture from 4 to 8.5. To examine the effects of amino acids on DDP synthetase activity, TABLE 111. EFFECT OF ADP AND AMP ON ASPARTOKINASE ACTIVITY Assay methods were the same as Table I. the coupled method was used at low substrate concentrations. That is, the concentrations of ASA and pyruvate were kept at 0.2 mm and 0.5 mm, respectively. As shown in Table V,. amino acids did not inhibit the activity. From the above mentioned results, the drawn conclusion was that DDP synthetase would not have regulatory site in the lysine biosynthesis of B. lactofermentum. ous addition of lysine and threonine concertedly inhibited the enzyme about 80% at 1 mm. When other amino acids were added, no effect was observed at 10 mm. As shown in Table III, ADP and AMP showed the inhibitory effect on aspartokinase. Results mentioned so far suggested that the aspartokinase from B. lactofermentum would be controlled by either threonine or lysine or both. DDP synthetase It had been reported that DDP synthetase from E. coli, the first specific enzyme in lysine TABLE IV. REQUIREMENTS FOR DDP SYNTHETASE ACTIVITY e) Complete system was as follows: Tris-HCl buffer (ph 8.0), 200 ƒêmoles; pyruvate, 1 ƒêmole; ASA, I µmole and enzyme in a total volume of 1ml.

98 0. TOSAKA and K. TAKINAMI of DDP synthetase reaction components was omitted, the successive DDP reductase reaction did not proceed as shown in Table VI. TABLE VI. REQUIREMENTS FOR DDP REDUCTASE ACTIVITY d) Complete system was as follows: Tris-HCl buffer (ph 7.5), 100,ƒÊmoles; pyruvate, 5 ƒêmoles; FIG. 1. Effect of ph and Lysine on DDP Synthetase. Assay methods were the same as those of Table IV except that the concentration of ASA and pyruvate were 0.2 mm and 0.5 mm, respectively. O, no addition; o, 10 miss of L-lysine added. TABLE V. EFFECT OF AMINO ACIDS ON DDP SYNTHETASE Assay methods were the same as those of Table IV, except that the concentration of ASA and pyruvate were 0.2 mm and 0.5 mm, respectively. L-Amino acids were added as indicated. ASA, 10 ƒêmoles; enzyme, 1.7 mg protein and NADPH, 0.1 mole in a total volume of 1 ml. TABLE VII. Assay methods EFFECT OF AMINO ACIDS ON DDP REDUCTASE were the same as those of Table VI, except that the amino acids indicated were added when NADPH was added. Amino acids were all in L-form and added as 10 mm concentration. Therefore, this indicated that DDP reductase was a specific enzyme of lysine biosynthesis in DDP reductase Repression of DDP reductase by lysine has been reported in S. aureus,12) while inhibition of this enzyme has not yet been reported. DDP reductase was partially purified from the sonic extract of the homoserine-requiring mutant of B. lactofermentum. This strain lacked homoserine dehydro genase whose contamination interfered the coupled assay of DDP synthetase as well as the assay of DDP reductase. When any one B. lactofermentum. Effect of amino acids on DDP reductase are shown in Table VII. Lysine and other amino acids of aspartate family did not inhibit the activity, but cysteine and alanine inhibited about 56 and 47 %, respectively, as compared with no addition. Result shows that DDP reductase was not a controlled site in lysine biosynthesis. AKAP synthetase N-Succinyl-ƒÃ-keto-ƒ -aminopimelate (AKAP)

Biosynthesis of L-Lysine and L-Threonine in Brevibacterium 99 has been proved to be an intermediate in the biosynthesis of lysine in E. coli by Gilvarg,13,14) TABLE IX. REQUIREMENTS FOR AKAP SYNTHETASE ACTIVITY who described the accumulation of AKAP by an auxotrophic mutant of E. coli. But he has never studied the properties and regulation of AKAP synthetase, since the substrate of this enzyme, 1 -piperideine-2,6-dicarboxylate, not be prepared because of its lability. could Works15), reported that the oxidation of L- diaminopimelate by L-amino acid oxidase brought about deamination of either a or r ami no group of diaminopimelate, and cyclation. In fact, 0.4,ƒÊmole of L-diaminopimelate was completely oxidized by 10 units of L-amino acid oxidase for 90 min. The deamination product was identified as 1 -piperideine-2,6-dicarboxylate by comparing the spectrum of its o-aminobenzaldehyde in neutral and acidic ph regions with the data of a) Complete system was as follows: Tris-HCl buffer (ph 7.5), 80 ƒêmoles; DAP, 0.4 smote; L-amino acid oxidase, 10 units; catalase, 60 units; succinyl-coa or acetyl-coa, 0.5 smote; KCl, 35 ƒêmoles and 1.7 mg of enzyme in a total volume of 1 ml. TABLE X. EFFECT OF LYSINE ON AKAP SYNTHETASE Assay methods were the same as the complete system of Table IX. Gilvarg. Requirements for 1 -piperideine-2,6-dicarboxylate formation are shown in Table VIII. The standard assay of AKAP synthetase was described in METHODS. Table IX shows the requirements for AKAP synthetase. This reaction required all components for 1 -piperi deine-2,6-dicarboxylate formation and suc cinyl-coa or acetyl-coa. AcyI donor for the reaction was reported to be succinyl-coa in E. coli, Aerobacter aeruginosa and Serratia marcescens.26) 181 In B. lactofermentum, either acetyl-coa or succinyl- TABLE VIII. REQUIREMENTS FOR 1 -PIPERIDEINE- 2,6-DICARBOXYLATE FORMATION Reaction mixture was incubated at 37 C for 90 min. After the reaction was stopped by the addition of 1 ml of I N HCl, 10 ƒêg of o-aminobenzaldehyde was added. After standing for 50 min at room temperature, the mixture was centrifuged at 3000 rpm for 10 min. Supernatant was measured at 300 nm. a) Complete system was as follows: Tris-HCl buffer (ph 7.5), 80 ƒêmoles; DAP, 0.8 smote; L-amino acid oxidase, 10 units and catalase 60 units in a total volume of I ml. CoA served as a acyl-donor, and acetyl-coa was a better substrate than succinyl-coa for this reaction as shown in Table IX. Effect of lysine on AKAP synthetase is shown in Table X. Lysine neither inhibited nor promoted the activity. DISCUSSION The pathway of lysine biosynthesis in Brevibacterium seemed to be the same as that in E. coli, since all enzymes related to the pathway in E. coli were found also in B. lactofermentum. In addition to this finding, it was found that AKAP synthetase, which has never measured in any microorganisms, could be detected in B. lactofermentum. Stadtman et al. have made an interesting discovery that extracts of E. coli contains at least two different and separable aspartokinase. One enzyme is specifically and competitively inhibited by lysine and the other is specifically and competitively inhibited by threonine.

100 0. TOSAKA and K. TAKINAMI Moreover, there is evidence for the existence of the third aspartokinase which is specifically repressed by methionine.17 On the other hand, Paulus and Gray,"' Datta and Gest,19) Miyajima and Shiio,8) and Nakayama110) reported that aspartokinases in Bacillus polymixa, Bacillus subtilis, Rhodopseudomonas capsulatus, Brevibacterium flavum and Corynebacterium glutamicum were inhi bited only when lysine and threonine were added simultaneously. This type of inhibition is called multivalent or concerted feedback inhibition. In B. lactofermentum, aspartokinase was inhibited about 45 % by lysine or threonine at I mm, respectively. When threonine and lysine were simultaneously added, about 80 inhibition was observed at each 1 mm. Ac cordingly, it should be noted that aspartokinase in B. lactofermentum was inhibited by a single or simultaneous addition of lysine and threo nine, and this type of the regulation was different from those in E. coli and B. flavum. It has been reported that DDP synthetase from E. coli, the first specific enzyme of lysine biosynthesis, is strongly inhibited by lysine, while those from Bacillus subtilis, Coryne bacterium glutamicum, B. flavum or Strepto coccus faecalis are not. The present results indicated that DDP synthetase was not inhibited by lysine. Thus, it would be concluded that in B. lactofermentum DDP synthetase might not have significant regulatory site in lysine biosynthesis. Repression of DDP reductase by lysine has been reported in Staphylococcus aureus, while inhibition of this enzyme has not yet been reported. In B. lactofermentum, DDP re ductase was not inhibited by lysine but cysteine or alanine. From the point of metabolic interlock in B. lactofermentum, it was of interest to eluci date the regulation of DDP reductase by these amino acids which are not related to lysine biosynthesis. The reports on AKAP synthetase have never been made on microorganisms. In B. lacto fermentum, the measurement of AKAP synthe tase proved that this enzyme was one of the specific enzyme in lysine biosynthesis, and either acetyl-coa or succinyl-coa served as an acyl donor for the reaction. Consequently, the biosynthetic pathway of lysine in B. lactofermentum may be the same as that in E. coli, while the regulation in lysine biosynthesis in B. lactofermentum may be different from that in E. coli, Acknowledgement. The authors indepted to direc ter Drs. I. Ota, T. Shiro and H. Okada of the laborato ries for their encouragements. They also wish to thank Mr. Y. Yoshihara and Mr. H. Hirakawa for the skillful assistance. REFERENCES 1) C. Gilvarg, Abstacts of papers, 143th National Meeting of American Chemical Society, Los Angels, Calif., April, 1963, p. 46. 2) R. Miyajima, S. Otsuka and I. Shiio, J. Biochem., 63, 139 (1968). 3) R. Miyajima and I. Shiio, Agric. Biol. Chem., 34, 1275 (1970). 4) S. Black and N. Wright, J. Biol. Chem., 213, 27 (1955). 5) Y. Yugari and C. Gilvarg, Biochim. Biophys. Acta, 62, 612 (1962). 6) Y. Yugari and C. Gilvarg, J. Biol. Chem., 240, 4710 (1965). 7) O. H. Lowry, N. J. Rosebrough and R. J. Randal, ibid., 193, 265 (1951). 8) I. Shiio and R. Miyajima, J. Biochem., 65, 849 (1969). 9) L. A. Chasin and J. Szulmajster, Biochem. Bio phys. Res. Commun., 29, 648 (1967). 10) K. Nakayama, H. Tanaka, H. Hagino and S. Kinoshita, Agric. Biol. Chem., 30, 611 (1966). 11) D. P. Gilboe, J. D. Friede and L. M. Henderson, J. Bacteriol., 95, 856 (1968). 12) I. J. Barnes, A. Bondi and A. G. Moat, ibid., 99, 160 (1969). 13) C. Gilvarg, Biochim. Biophys. Acta, 24, 216 (1957). 14) C. Gilvarg, J. Biol. Chem., 234, 2955 (1959). 15) E. Works, Biochim. Biophys. Acta, 17,410 (1955). 16) K. Nakayama, H. Kase and S. Kinoshita, Agric. Biol. Chem., 34, 282 (1970). 17) E. R. Stadtman, G. N. Choen, S. B. Wieseudange and M. L. Hirsch, Compt. Rend., 236, 1342 (1954). 18) H. Paulus and E. Gray, J. Biol. Chem., 239, 4008 (1964). 19) P. Datta and H. Gest, Proc. Natl. Acad. Sci, U.S.A., 52,1004 (1964).