J. Biochem., 68, 859-866 (1970)
Homoserine Dehydrogenase Genetically Desensitized
to the Feedback Inhibition in Brevibacterium flavum
Isamu SHIIO, Ryuichi MIYAJIMA and Shigeru NAKAMORI The Central Research Laboratories of Ajinomoto Co ., Inc., Kawasaki
Received for publication, April 21, 1970
Homoserine dehydrogenase [L-homoserine : NADP oxidoreductase , EC 1. 1. 1. 3] from Brevibacterium flavum and from its mutants which were resistant to DL-ƒ¿-amino-ƒÀ-
hydroxyvaleric acid (AHV),* a threonine analogue, and which produced a large amount
,of threonine , were partially purified and their properties were compared with each other. AHV inhibited the parental enzyme, similarly to the end products , threonine and isoleucine. The types of the inhibition were competitive to both of the two sub strates. In the presence of AHV, homotropic cooperativity of NADPH was observed , as in the presence of threonine or isoleucine. In contrast to the parental enzyme, the mutant enzymes were scarcely inhibited by these amino acids. Concentration of threonine giving 50%'o inhibition was above 80 mm with the mutant enzymes while 0.06 mm with the parental enzyme. On the other hand, the inhibition by the reaction products, homoserine and NADP+, were of similar degree with the two enzymes. In the absence of KCI, homoserine dehydrogenase of the parental strain was rapidly inactivated, which was protected by the addition of threonine, while the mutant enzymes were stable without both KCl and threonine. Molecular weight of homoserine dehydrogenase, which was determined to be about 2.5•~105 by the gel filtration experiments, was not different between the parental and
the mutant strains. K,s for the substrates, aspartic-ƒÀ-semialdehyde (ASA)* and NADPH, as well as
optimum pH for the reaction were similar with these enzymes. Repression of homoserine dehydrogenase by methionine was similarly observed with the two mutants. Aspartate kinase [EC 2.7.2.4] from one of the mutant strains was concertedly inhibited by lysine plus threonine, similarly to that from the parental strain. From the gel filtration experiments, it was confirmed that aspartate kinase and homoserine dehydrogenase of B. flavum were not aggregated with each other in contrast to threonine-sensitive enzymes of Escherichia coli. It is likely from these results that resistance of the mutants to AHV as well as
* Abbreviations used were AHV for ƒ¿-amino-ƒÀ-hydroxyvaleric acid and ASA for aspartic-ƒÀ-semialdehyde .
Vol. 68, No. 6, 1970 859 860 1. SHIM, R. MIYAJIMA and S. NAKAMORT
threonine overproduction in the mutants are due to the lack of the feedback inhibi tion in the mutant homoserine dehydrogenase. This supports the previous suggestion that homoserine dehydrogenase may be the primary control site in the threonine bio- synthesis in B. flavum.
In the previous paper, it was shown that some Organisms-B. flavum No. 2247 (ATCC mutants resistant to a threonine analogue, 14067), strain B-183, a mutant resistant to 5 AHV, derived from B. flavum excreted a large mg/mi of AHV from strain No. 2247, and amount of threonine in the culture medium strain BB-69, a mutant resistant to higher con (1 ). In the threonine biosynthesis of B. flavum, centration of AHV (8 mg/ml) from strain B- aspartate kinase [EC 2. 7. 2. 4] is concertedly 183 (1), were used for preparation of homo inhibited by lysine plus threonine (2), homo serine dehydrogenase. serine dehydrogenase [EC 1. 1. 1. 3] is strongly, Preparation of Homoserine Dehydrogenase- and homoserine kinase [EC 2. 7. 1. 39] is weakly B. flavum B-183 and strain BB-69 were cultured susceptible to feedback inhibition by threonine at 30•Ž for 27 hr and 35 hr, respectively, in (3, 4 ). Moreover, homoserine dehydrogenase glucose-salts medium containing 30 ƒÊg/liter of as well as homoserine kinase is repressed by biotin (7 ), and homoserine dehydrogenases methionine (5 ). However, main control site were partially purified about 23- and 26-fold, of the threonine biosynthesis seems to be respectively, by the same methods as used homoserine dehydrogenase, since it is more with the parental strain (4). The enzyme
strongly inhibited by low concentration of preparations thus obtained were used through- threonine rather than the other two enzymes out this experiments unless otherwise cited. ( 2-4 ). Therefore, the overproduction of thre Assay of Homoserine Dehydrogenase-Homo onine in the mutants strongly suggests a genetic serine dehydrogenase activity was measured desensitization of homoserine dehydrogenase by the same method as described previously to the feedback inhibition. (4). Components of the standard assay condi Thus, in this paper, some properties of tions are as follows : 100 mm potassium phos partially purified homoserine dehydrogenases phate buffer, pH 7.0, 0.1 mm NADPH, 1.33 mm from B. flavum No. 2247 and its mutants (1) ASA (as L-form) and enzyme in a total volume resistant to AHV, which produced a large of 1.5 ml. In the case of inhibition test, con amount of threonine, were compared with each centration of ASA used was 0.2 mM. other. As a result, it was confirmed that Protein concentration was measured by homoserine dehydrogenases from the mutants the method of Lowry et al. (8). became insensitive to the feedback inhibitors. Purification and Assay of Aspartate Kinase-Aspartate kinase from strain BB-69 as
MATERIALS AND METHODS well as strain No. 2247 was partially purified and was assayed by the same methods as. Chemicals-Amino acids used were all in described previously (2). L-form unless otherwise cited and purchased from Sigma, Calbiochem, or NBCo. DL-AHV, RESULTS NADP+ and NADPH were obtained from Sigma. ASA was prepared by ozonolysis of DL-allyl General Properties of Homoserine Dehy glycine (Sigma) by the method of Black and drogenases from AHV Resistant Mutants-Spe Wright ( 6). Markers used in gel filtration cific homoserine dehydrogenase activities of experiments were blue dextran (Pharmacia), the crude extracts from AHV resistant mutants, glutamate dehydrogenase [EC 1. 4. 1.3] of beef strain B-183 and BB-69 were 0.471 and 0.481, liver (Boehringer), alcohol dehydrogenase [EC respectively, similar to that of the parental 1. 1. 1. 1] from yeast (Boehringer), and cyto strain which was 0.452. Homoserine dehy chrome c from yeast (Boehringer). drogenases from strain B-183 and BB-69 were
J. Biochem. DESENSITIZED HOMOSERINE DEHYDROGENASE 861
then partially purified about 23-fold and 26-fold with total yield of 41% and 49%, respectively. Partially purified preparations described in the above contained little amount of ASA dehy drogenase [EC 1.2.1.11], dihydrodipicolinate synthetase, and NADPH oxidase system as in the case of the parental enzyme preparations (4). The enzymes from both mutants were active in wide pH range, and optimum pH of the reaction was 6.0 with potassium phosphate buffer, the same as for the parental enzyme (4). Similarly to the parental enzyme, double reciprocal plots of the reaction rate against concentrations of one substrate at various fixed
levels of concentration of another substrate, Fig. 1. Effect of products, NADP+ and homoserine, gave straight lines and met at a point on the on the forward reaction of homoserine dehydro base line with enzymes from the mutants. genases. 5-6ƒÊg protein/ml was used under the K,„s for ASA of homoserine dehydrogenase standard assay conditions except that the concentra from strain No. 2247, B-183, and BB-69 were tion of ASA used was 0.2 mm. --, refined curve of homoserine on the enzyme from strain No. 2247 1.90 x 10-4 M, 3.57•~10-4M, and 3.77•~10-4 M, (see ref. (4)); -, refined curve of NADP+ on the respectively, and those for NADPH were 3.74 x enzyme from strain No. 2247 (see ref. (4)); •~ , homo 10-5 M, 4.00 x 10-5 M, and 5.75•~10-5M, respec serine on the enzyme from strain B-183; , homo tively. serine on the enzyme from strain BB-69; C), NADP+
As shown in Fig. 1, the reaction products, on the enzyme from strain B-183; •œ , NADP+ on the
homoserine and NADP+, inhibited the forward enzyme from strain BB-69.
Fig. 2. Effect of threonine or isoleucine on homoserine dehydrogenases from
the parental and the mutant strains. The reactions were performed under
the standard assay conditions except that the concentration of ASA used was
0.2 mm. -•œ-, threonine with the enzyme of strain No. 2247 (6ƒÊg protein) ; -- •œ -- , isoleucine with the enzyme of strain No. 2247 (6ƒÊg protein) ; -•~ ,
threonine with the enzyme of strain B-183 (5ƒÊg protein) ; -- •~ --, isoleucine
with the enzyme of strain B-183 (5 ƒÊg protein) ; -•ü -, threonine with the
enzyme of strain BB-69 (6ƒÊg protein) ; --•ü --, isoleucine with the enzyme
of strain BB-69 (6 ƒÊg protein).
Vol. 68, No. 6, 1970 862 I. SHIIO, R. MIYAJIMA and S. NAKAMORI reaction in both mutant enzymes as in the parental enzyme (4). Effect of End Products, Threonine and Isoleucine, on Homoserine Dehydrogenase-Ef fects of threonine and isoleucine on the mutant enzymes were compared with those on the parental enzyme. The results are shown in Fig. 2, which indicate that the mutant en zymes are almost insensitive to the actions of these end products in contrast to the parental enzyme. Thus, 50% inhibition by threonine took place at 0.06 mm with the parental en zyme, whereas at more than 80 mm with the mutant enzymes. Thus, susceptibility of the mutant enzyme to the inhibition by threonine was less than one thousandth of that of the parental enzyme. Fig. 3. Effect of AHV on homoserine dehydrogenase Since these enzyme preparations are not activity of strain No. 2247 and BB-69. 5-6 ƒÊg pro completely pure, it is possible that above men tein/ml of homoserine dehydrogenases from BB-69 tioned differences in the effect of threonine or strain No. 2247 was used under the standard and isoleucine between these enzymes may be assay conditions except that the concentration of due to impurities in the enzyme preparations. ASA used was 0.2mM. •ü, strain No. 2247; •œ
However, it was not the case, because effects strain BB-69. of threonine and isoleucine on a mixture of these enzymes were simply additive. Any differences in the inhibition by thre onine between strains B-183 and BB-69 were not observed, although the latter strain was more resistant to AHV and accumulated larger amount of threonine than the former (1). Isoleucine did not inhibit the mutant en zymes at the concentrations used (up to 40 mm). Effect of Threonine Analogue, AHV, on Homoserine Dehydrogenase-As shown in Fig. 3, AHV inhibited the parental enzyme more strongly than isoleucine but much more weakly than threonine, and concentrations of L-AHV, Fig. 4. Double reciprocal plots of the reaction rate
L-isoleucine and L-threonine giving 50% inhibi against ASA (a) or NADPH (b) concentrations in tion were 1.25 mm, 4.2 mm and 0.06 mm, re the presence or absence of AHV with the parental spectively. Effects of AHV were similar to enzyme. •œ, no inhibitor; 3mM DL-AHV added. those of isoleucine (4 ). Namely, the inhibi The other conditions were the same as those of the tion curve became sigmoidal, and the inhibi standard assay. tion types by AHV were competitive with both substrates as shown in Fig. 4. tropic cooperativity of NADPH. Double reciprocal plots of the reaction rate Effects of AHV on homoserine dehydro against ASA concentrations in the presence or genases from both mutants are shown in Fig. absence of AHV gave straight lines, while 3, which indicates that AHV did not inhibit those against NADPH concentrations in the the enzymes at the concentrations tested (up presence of AHV bent upward, showing homo- to 10 mm).
J. Biochem. DESENSITIZED HOMOSERINE DEHYDROGENASE 863
Stability of Homoserine Dehydrogenase-As when KC1 was removed, just as the parental described in the previous paper (4 ), homoserine enzyme was in the presence of 1 mm threonine, dehydrogenase of the parental strain became as shown in Fig. 5. The addition of threonine very unstable when KCl in the enzyme prepa to the mutant enzymes did not show further ration was removed, and it was stabilized by protective effect. the addition of threonine. On the other hand, Repression of Homoserine Dehydrogenases the mutant enzymes were fairly stable even by Methionine-Homoserine dehydrogenase of the strain No. 2247 is susceptible to repression by methionine (5) as other bacteria and yeast (9 ). Repressive effect of methionine on homo serine dehydrogenase of the threorine produc ing mutants resistant to AHV was studied. Sonic extracts obtained from cells grown in glucose-salts medium (7) containing various levels of methionine were treated with 4 vol umes of saturated ammonium sulfate in order to remove low molecular weight substances. The preparations thus obtained were assayed for homoserine dehydrogenase activity under the standard conditions. The results are shown in Table I, which indicate that homoserine dehydrogenases of the mutants were repressed by methionine, as well as that of the parental strain. Significant growth inhibition was ob-
Fig. 5. Stability of homoserine dehydrogenases from served with the strain BB-69 in the presence the parental and mutant strains. For desalting, each of higher concentration of methionine. enzyme preparation was passed through Sephadex Aspartate Kinase from Strain BB-69-As G-50 column with 0.05M phosphate buffer, pH 7.0, described in the previous paper ( 2 ), aspartate and the activities were measured under the standard kinase, the first step common enzyme for the assay conditions. After stored in ice for 2, 4, and threonine and lysine biosynthesis, is controlled 6hr, with or without threonine (1 mM), the activities through the concerted inhibition by lysine plus were assayed again. The percentage of residual ac threonine in B. flavum, although the inhibi tivities were shown in the figure. i,•œ strain No. tion is weaker than that of homoserine dehy 2247 without threonine ;•ü strain No. 2247 with threonine ; . _ , strain B-183 without threonine ; •~ , drogenase (4 ). Therefore, aspartate kinase strain BB-69 without threonine. from strain BB-69 as well as from strain No.
TABLE 1. Effect of methionine on homoserine dehydrogenase formation. B. flavour No. 2247, strain B-183 and strain BB-69 were cultured at 30°C for 40 hr, 40 hr and 48 hr, respectively, in glucose-salts medium ( 7 containing various levels of methionine. Homoserine dehydrogenase activities of the cells were measured under the standard assay conditions.
1) Optical density at 562 my with 26-fold dilution. 2) ƒÊmoles of NADPH oxidazed,'min/mg protein.
Vol. 68, No. 6, 1970 864 I. SHIIO, R. MIYAJIMA and S. NAKAMORI
TABLE II. Effect of amino acids on aspartate kinases from the parental and the mutant strains. Aspar tate kinase of the strain BB-69 as well as the parental strain was partially purified and the activities in the presence or absence of amino acid(s) were assayed under the standard conditions except that the con centration of aspartate used was 10 mm, as described previously (2).
2247 was partially purified by the same method as described previously (2), and their proper- ties were compared with each other. In con- sequence, Kms for the substrates, aspartate and ATP, were 53 mm and 5.3 mm, respectively, similar to those for the parental enzyme (50 and 8.1 mm, respectively). While low concen tration of threonine or lysine alone showed little inhibitory effect on aspartate kinase from strain BB-69, simultaneous addition of lysine and threonine even at low concentrations caused strong inhibition of the mutant enzyme (Table II). As shown in the table, susceptibility to the concerted inhibition by lysine plus threonine was not altered in the mutant enzyme. Fur thermore, AHV itself scarcely inhibited the parental enzyme, and AHV plus lysine did not inhibit the activity significantly. Gel Filtration Experiments-In order to elu Fig. 6. Elution pattern of the parental and the cidate difference in the molecular weight be mutant homoserine dehydrogenases from Sephadex
G-200. Sephadex G-200 column (2.2 x 48 cm) was tween the parental and the mutant homoserine equilibrated with 0.1M phosphate buffer, pH 7.0, dehydrogenase, a gel filtration experiment was containing 0.4M ammonium sulfate. The elution performed using Sephadex G-200 column equi buffer was the same as that used in equilibration of librated with 0.1 M potassium phosphate buffer, the column and the elution was carried out at 0- pH 7.0, containing 0.4 M ammonium sulfate. 3°C. Flow rate was 8 ml/hr. The markers used were At the same time, aspartate kinase purified blue dextran (MW 2 X 106), glutamate dehydrogenase of beef liver (MW 3 x 105 (10)), alcohol dehydro tivities were assayed under the standard conditions genase from yeast (MW 1.5•~105), cytochrome c from as well as those containing 10 mm threonine. The yeast (MW 1.26•~104), and aspartate kinase from B. numbers in the figure represent glutamate dehydro flavum No. 2247 (MW about 8•~104) (12). Homo genase for 1, homoserine dehydrogenases from the serine dehydrogenases from the mutant (BB-69) and parental and the mutant strains for 2, alcohol de the parental strains were passed through the column hydrogenase for 3, aspartate kinase for 4, and .cyto at the same time with other markers, and their ac- chrome c for 5.
J. Biochem. DESENSITIZED HOMOSERINE DEHYDROGENASE 865• from the parental strain was used as one of Whereas a higher concentration of isoleucine the markers in order to study the relationship as well as AHV inhibited the parental enzyme between aspartate kinase and homoserine dehy almost completely, inhibition by them was not drogenase, since threonine-sensitive aspartate observed with the mutant enzymes at the con kinase and homoserine dehydrogenase in E. centrations tested. coli have been shown to aggregate with each Furthermore, whereas homoserine dehy other to form a single protein (10). As shown drogenase from strain No. 2247 became very in Fig. 6, homoserine dehydrogenases from unstable when desalted, and was protected from both parental and mutant (BB-69) strains were such inactivation by the addition of threonine, eluted at the same point and aspartate kinase enzymes from both two mutants were fairly was eluted at a different point from homo stable without threonine, even after desalting, serine dehydrogenases. From Fig. 6, the mo and the degree of stability was just the same lecular weight of homoserine dehydrogenase of as that of the parental enzyme protected by B. flavum was calculated to be about 2.5 x 105. the addition of 1 mm threonine. Another gel filtration experiment was carried On the other hand, homoserine dehydro out at a low concentration of inorganic salts, genase levels in the cell extracts, optimum using Sephadex G-200 column equilibrated with pH of the catalyzed reaction, K,,, values for 0.05M potassium phosphate buffer, pH 7.0, the substrates, and the inhibition by the reac containing 0.001M threonine as a stabilizer. tion products were almost identical with both However, the results were almost the same as the parental and mutant enzymes. Further- described above. more, repression by methionine of homoserine dehydrogenase were observed with the mutant strains, as with the parent. Also, molecular DISCUSSION weight of homoserine dehydrogenase was not As reported previously (1), growth of B. different between the parental strain and the flavum No. 2247 is strongly inhibited by thre mutant, BB-69. onine analogue, AHV, and is recovered spe In addition to the feedback inhibition of cifically by the further addition of threonine. homoserine dehydrogenase by threonine, as This growth inhibition can be explained by partate kinase is concertedly inhibited by lysine the present results that homoserine dehydro plus threonine (2, 3) and homoserine kinase genase, the primary control site of the thre is weakly inhibited by threonine (3), in the onine biosynthesis in B. flavum, is significantly threonine biosynthetic pathway of B. flavum. inhibited by AHV as well as threonine and Aspartate kinase of the AHV resistant mutant, isoleucine. Several inhibitory properties of strain BB-69, was partially purified and its AHV tested were rather similar to those of properties were compared with that of the isoleucine than those of threonine. parental strain. In the results, inhibition by It has been also reported that some mutants lysine plus threonine or threonine alone, and resistant to AHV derived from B. flavum No. K,, values for the substrates, were almost simi 2247 accumulated a large amount of threonine lar to those of the parental enzyme. in the culture media (1). As will be reported on homoserine kinase In the present study, remarkable differ elsewhere, threonine inhibition of homoserine ences in the sensitivity of homoserine dehy kinase was not altered by the mutation. drogenate to the allosteric effectors have been In E. coli, it has been reported that both found between the parental and the mutant threonine-sensitive aspartate kinase and thre strains. Threonine concentration giving 50% onine-sensitive homoserine dehydrogenase were inhibition was more than 80 mM with the inhibited by AHV (11) and that, in revertants mutant enzymes, whereas 0.06 mM with the from lysine-sensitive aspartate kinase defective mutant of E. coli, both enzymes became lesss parental enzyme. Thus, the mutant enzymes were at least 1,000-fold insensitive to the in sensitive to threonine, simultaneously (10 ). hibition by threonine than the parental enzyme. This is in good agreement with the finding.
Vol. 68, No. 6, 1970 866 I. SHIIO, R. MIYAJIMA and S. NAKAMORI
that the two enzymes mentioned above ag Therefore, threonine overproduction in AHV gregate with each other (10). On the other resistant mutants suggests that intracellular hand, aspartate kinase was found to consist levels of lysine might be very low under these of a different protein from homoserine dehy conditions. In this point, it should be noted drogenase in B. flavum, since it was separated that, since the levels of dihydrodipicolinate from the latter by gel filtration. As expected, synthetase and homoserine dehydrogenase in desensitization of homoserine dehydrogenase B. flavum, which situate at the branch point to inhibition by threonine was not accompanied on the lysine and threonine biosynthetic path by an alteration of the effect of threonine on way, are about 1: 15 (13 ), ASA, an inter aspartate kinase in B, flavum, in contrast to mediate at the branch point, will be predom those in E. coli. inantly used for threonine biosynthesis, when In conclusion, main difference between threonine thus produced does not control the strain No. 2247 and the threonine producing activity of homoserine dehydrogenase, as with mutants resistant to AHV seems to be desen the mutants. Therefore, it is likely that the sitization of homoserine dehydrogenase to the intracellular levels of lysine of the mutants inhibition by allosteric inhibitors, threonine became limiting in the mutants. and isoleucine as well as AHV, and the in- The authors are indebted to Director K. Akino and creased stability of homoserine dehydrogenase Dr. T. Tsunoda of their laboratories for the encour in the absence of protectors, threonine and agement during the course of this study. inorganic salts. As described previously (4), the inhibition REFERENCES by threonine of the parental homoserine dehy drogenase in B. flavum is due to an indirect 1. I. Shiio and S. Nakamori, Agr. Biol. Chem., 43, interaction caused by binding of threonine to 448 (1970). an allosteric site of the enzyme, and when 2. I. Shiio and R. Miyajima, J. Biochem., 65, 849 threonine binds to the site, the stability of the (1969). enzyme is increased, simultaneously. There- 3. R. Miyajima, S. Otsuka and I. Shiio, J. Biochem., fore, conformation of homoserine dehydro 63, 139 (1968). 4. R. Miyajima and I. Shiio, J. Biochem., 68, 311 genase would have been genetically altered in (1970). the mutant strains, not only to increase the 5. R. Miyajima, S. Otsuka and 1. Shiio, Abstract stability of the enzyme but also to desensitize of the 39th General Meeting of Japanese Agri it to the inhibition by threonine. cultural Chemistry, p. 272 (1968) (in Japanese). The fact that desensitization of homoserine 6. S. Black and N. Wright, J. Biol. Chem., 213, 39 dehydrogenase to the threonine inhibition alone (1955). caused the production of a large amount of 7. I. Shiio, S. Otsuka and T. Tsunoda, J. Biochem., threonine in the culture medium is in good 46, 1303 (1959). agreement with the previous suggestion that 8. O.H. Lowry, N.J. Rosebrough and R.J. Randal, homoserine dehydrogenase is the primary con J. Biol. Chem., 193, 265 (1951). 9. E.R. Stadtman, Adv. in Enzymology, 28, 72 (1966). trol site of the threonine biosynthetic pathway 10. J-C. Patte, P. Truffa-Bachi and G.N. Cohen, Bio in B. flavum (4 ). chim. Biophys. Acta, 128, 426 (1966). As already mentioned, aspartate kinase, 11. G.N. Cohen and J-C. Patte, Cold Spring Harbor the first common enzyme of the lysine and Symposia on Quantitative Biology, 28, 513 (1963). threonine biosynthesis, is susceptible to con 12. I. Shiio, R. Miyajima and K. Sano, J. Biochem., certed feedback inhibition by lysine plus thre 68, 701 (1970). onine in both the parental and the mutant 13. R. Miyajima and I. Shiio, Agr. Biol. Chem., 34. strains. This feedback control is actually strong 1275 (1970). enough to prevent lysine overproduction (12 ).
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