Regulatory Properties of L-Methionine Biosynthesis. Homoserine- O-transsuccinylase*

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1 Agric. Biol. Chem., 46 (1), 57-63, Regulatory Properties of L-Methionine Biosynthesis in Obligate Methylotroph OM33: Role of Homoserine- O-transsuccinylase* Yasushi Morinaga,** Yoshiki Tani and Hideaki Yamada Department of Agricultural Chemistry, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received June ll, 1981 A cell-free extract of obligate methylotroph strain OM33 catalyzed the formation of O- succinyl-l-homoserine from L-homoserine and succinyl-coa, while the corresponding homoserine derivative from acetyl-coa was scarcely formed. These results indicate that the acylation of l- homoserine, the initial step of L-methionine biosynthesis, was catalyzed by homoserine-otranssuccinylase. In this bacterium, homoserine-o-transsuccinylase was subject to strict feedback inhibition by S-adenosyl-L-methionine (SAM). On the other hand, the enzyme of an ethionineresistant mutant OE 120 derived from strain OM33, was scarcely affected by SAM. These observations suggest the important role of homoserine-o-transsuccinylase in the biosynthesis of l- methionine. In the previous paper,1} we reported that ethionine-resistant mutants derived from an obligate methylotroph, strain OM 33, overproduced L-methionine in a methanolmedium. For the typical mutant, strain OE 120, it was suggested that an enzyme or enzymes related to L-methionine biosynthesis were modified by mutation so as to lose sensitivity to L-methionine or L-methionine derivatives. In this paper, we report on the regulation of L-methionine biosynthesis in obligate methylotroph strain OM33. We found that homoserine-o-transsuccinylase, the first enzyme in L-methionine biosynthesis, is strictly regulated by S-adenosyl-L-methionine (SAM) and the enzyme of mutant OE 120 lost the sensitivity. Although it has been reported that the enzyme concerned in acylation of l- homoserine is regulated by L-methionine and/or SAM in some microorganisms,2~7) nothing is known about the pathway or regulation of L-methionine biosynthesis in methanol-utilizing bacteria. * Formation of L-Methionine by Methanol-utilizing Bacteria. Part II. ** On leave from the Central Research Laboratories of Ajinomoto Co., Kawasaki, Japan. MATERIALS AND METHODS Microorganisms. Obligate methylotroph strain OM33, which is a Gram-negative rod and assimilates methanol via the ribulose monophosphate pathway, was isolated from soil and characterized as described previously.x) An ethionine-resistant mutant, strain OE 120, was derived from strain OM33 by treatment with N-methyl-TV'-nitro- TV-nitrosoguanidine.1* Microorganisms were cultured in a methanol-salts medium (PC medium)1} containing various amounts of L-methionine as described elsewhere in the text. Cultivation was carried out at 28 C for 40hr with shaking. Preparation of cell-free extracts. Cell-free extracts were prepared essentially as described previously.1} However, potassium phosphate buffer, ph 7.5, containing 10mM ethylene diaminetetraacetic acid, 0. 1 him /?-mercaptopropionate and 0.05 mmpyridoxal 5'-phosphate, was used throughout the" procedures of washing, suspending and disrupting cells. In some cases, 5ml of the extract was dialyzed against 2 liters of the same buffer at 4 C for 18 hr. Enzymeassays. Homoserine-O-transsuccinylase activity was assayed by measuring the L-homoserine dependent release of free CoA from succinyl-coa according to the method of Lee et al.8) The reaction mixture contained 50 //mol Tris-HCl buffer, ph 7.5, 1.25 //mol L-homoserine, 0.14 fimol succinyl-coa and dialyzed extract in a total volume of 0.5ml. The reaction was started by addition of the extract. After incubation at 30 C for 10 min, the reaction was stopped by the addition of 1ml of cold

58 Y. Morinaga, Y. Tani and H. Yamada ethanol. Then, 2.5 ml of 0.08 m potassium phosphate buffer, ph 8.0, and 0.02ml of 0.01m 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB) (in 0.

2 58 Y. Morinaga, Y. Tani and H. Yamada ethanol. Then, 2.5 ml of 0.08 m potassium phosphate buffer, ph 8.0, and 0.02ml of 0.01m 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB) (in 0. 1 m potassium phosphate buffer* ph 7.0) were added. After standing for 10 min at roomtemperature, the optical density at 412nmwas measured. The amount of free CoAreleased was calculated from the molar extinction coefficient of DTNB (1.36x 104).9) Blanks were run omitting L-homoserine from the complete system. Serine-O-transacetylase activity was assayed by the same method as that for homoserine-0-transsuccinylase except that 1.25 /xmol of L-serine and 0.14 /miol of acetyl- CoAwere added instead of L-homoserine and succinyl- CoA, respectively. O-Acetylserine sulfhydrylase activity was assayed by modification of the method of Yamagata.10) The reaction mixture contained 50 fimol Tris-HCl buffer, ph 7.8, 0.1 /imol pyridoxal 5'-phosphate, 10 ^wrnol O-acetyl- L-serine, 1.6 /unol Na2S and cell-free extract in a total volume of 0.5ml. The blanks contained no 0-acetyl-Lserine. The reaction was carried with a rubber stopper. After out in a test tube sealed incubation at 30 C for 10 min, the reaction was stopped by the addition of 0.05ml of cold 1.5m trichloroacetic acid. Then, watersaturated N2 gas was bubbled through the reaction mixture for 3 min in order to remove the residual H2S. To a 0.1ml aliquot of the reaction mixture, 2.5ml of 0.08m potassium phosphate buffer, ph 8.0, and 0.016ml of 0.01 m DTNBwere added, and the amount of L-cysteine formed was determined. Protein was determined according to the method of Lowryet al.n) Chemicals. Succinyl-CoA and acetyl-coa were prepared by the methods of Simon and Shemin,12) and Wilson,13) respectively. O-Acetyl-L-serine was synthesized according to the method of Nagai and Flavin.14) Other chemicals were of the purest grade available commercially. RESULTS Homoserine-O-transsuccinylase of obligate methylotroph OM33 a) Acyl donor for acylation of l- homoserine. Acylation of L-homoserine was examined with the dialyzed extract prepared from strain OM33 cells grown on methanol. As shown in Fig. 1, release of free CoA occurred significantly when succinyl-coa was added as the acyl donor. In the presence of acetyl-coa instead of succinyl-coa, slow development of sulfhydryl titer of free CoA occurred. However, the reactivity of acetyl- CoA was less than one-eighth of that of succinyl-coa. This indicates that a better acyl "o / 4 c / -6- / ~& / CD lu' <2-J o J r J Incubation time(min) Fig. 1. Acylation of L-Homoserine by Cell-free Extract of Obligate Methylotroph OM33. The complete reaction mixture contained 50 fimol Tris- HC1, ph 7.5, 1.25 /imol L-homoserine, 0.14 /xmol succinyl- CoA and enzyme (186/xg protein) in a total volume of 0.5 ml. #, complete reaction mixture; O, acetyl-coa was added instead of succinyl-coa; A, no acyl-coa added. donor for acylation of L-homoserine in this bacterium is succinyl-coa. In the complete reaction system, the increase of the sulfhydryl titer was linear up to 10 min (Fig. 1). The relationship between enzyme concentration and reaction velocity was proportional up to 0.5mg protein in the system. b) Effect ofl-methionine on the formation of homoserine-o-transsuccinylase. Strains OM 33 and OE 120 were cultured in a methanolmedium supplemented with various concentrations of L-methionine, and the levels of homoserine-o-transsuccinylasewereexamined (Table I). Even in the cells of both strains grown with 10mML-methionine, only a slight decrease in the enzyme activity These results suggest that the was observed. formation of homoserine-o-transsuccinylase in this bacterium is not regulated by the feedback repression. c) Effect of L-methionine and SAMon homoserine-o-transsuccinylase activity. Table II shows the effect of L-methionine, l- ethionine and SAM on the activity of homoserine-o-transsuccinylase in the dialyzed extracts prepared from the cells of OM33 and OE 120. L-Methionine and L-ethionine in-

3 L-Methionine Biosynthesis in Obligate Methylotroph 59 Table I. Effect of l-methionine on Synthesis of Homoserine-O- TRANSSUCCINYLASE Strains OM33 and OE 120 were grown for 40hr in the presence of L-methionine as indicated. The activity of homoserine-0-transsuccinylase was determined as described in Materials and Methods. nmol/min/mg protein. Table II. Effect of l-methionine and SAMon Activities of Homoserine- O-TRANSSUCCINYLASE Except for the addition of amino acids, the enzyme assay was carried out as described in Materials and Methods. Table III. Effect of Amino Acids on Activity of Homoserine- O-transsuccinylase The activities of the enzymein the presence or absence of the amino acids indicated were assayed as described in Materials and Methods. The concentrations of l- homoserine and succinyl-coa used were 2.5 and 0.22 mm, respectively. The activities are expressed relative to the value obtained with no addition of an amino acid. binationwasnot observedwiththe cell-free extracts of both strains. Table III showsthe effect of amino acids related to the aspartate family other than L-methionine and SAM on homoserine-0-transsuccinylase activity. Thoughno significant effect was observed with these amino acids, 0-succinyl-L-homoserine, one of the reaction products, inhibited the activity by 30% at 10mM. The activities of homoserine-0-transacetylase in Brevibacterium flavum6) and Corynebacteriumglutamicum1] are knownto be inhibited by 0-acetylhomoserine. d) Properties of homoserine-0-transsucinylase in ethionine-resistant mutant OE 120. Doublereciprocal plots of the reaction rate of homoserine-o-transsuccmylase the presence or absence of SAM against l- homoserine (or succinyl-coa) concentration are shown in Fig. 2. SAMis a non-competitive type inhibitor of the enzyme from OM33 with respect to both substrates. TheKi value for SAMwas calculated to be 0.04mMat the concentration of succinyl-coa of 0.28mM. Meanwhile,with the enzymefrom OE120, the

4 60 Y. Morinaga, Y. Tani and H. Yamada -1' r r ~ A / B, E06å f - y /Homoserine(mM) 1 /Succinyl-CoA(mM) /Homoserine(mM) 1 /Succinyt-CoA(mM) Fig. 2. Effect of Concentration of L-Homoserine or Succinyl-CoA on Homoserine-O-transsuccinylase. The enzymeactivities in the dialyzed extracts prepared from cells of OM33 and OE120 were assayed by the method described in Materials and Methods except that 0.063mM SAMwas added (#) or not added (O) to the reaction mixture. A, enzyme from strain OM33, succinyl- CoA 0.28mM; B, enzyme from strain OM 33, l- homoserine 2.5mM; C, enzyme from strain OE 120, succinyl-coa 0.28 mm; D, enzyme from strain OE 120, l- homoserine 2.5 mm. Ki value for SAMincreased to more than 10 times as much as that of OM33. On the contrary, the enzyme of OM33 and OE 120 showedthe same affinity to substrates. At the concentration of succinyl-coa of 0.28 him, the apparent Kmvalues of the enzyme from both strains for L-homoserine were 0.9 mm,while at 2.5 mml-homoserine, the corresponding parameters for succinyl-coa were 0. 1 mm. SAM probably attaches to the allosteric site of the enzyme, because it inhibits the enzyme activity non-competitively. In the mutant cells, the affinity of the enzyme to SAMdecreased but the affinities to substrates did not change. This indicates that the allosteric site, where SAM attaches, of the enzymeprotein might be altered by mutation. O.o /»> <2\ <S) Incubation time(min) Fig. 3. Time Course of Free CoA Liberation in the Serine-0-transacetylase Reaction in Obligate Methylotroph OM 33. The complete reaction mixture contained 50 /imol Tris- HC1, ph 7.5, 1.25 /tfnol L-serine, 0.14 jumol acetyl- CoA and enzyme (186jig protein) in a total volume of 0.5ml. #, complete reaction mixture; O, succinyl- CoAwas added instead of acetyl-coa; A, no acyl-coa was added. Regulation of enzymes concerned with.lcysteine biosynthesis by L-methionine It is knownthat the sulfur in L-methionine is derived from L-cysteine in many microorganisms. Therefore, it is necessary to investigate the effect of L-methionine on the l- cysteine biosynthesis in order to elucidate the overall regulation mechanism of L-methionine biosynthesis. The participation of the two enzymes, i.e., serine-o-transacetylase and O- acetylserine (OAS) sulfhydrylase, in L-cysteine biosynthesis has already been established in Escherichia coli,15) Salmonella typhimurium,15) Desulfovibrio vulgaris,16) yeast17) and spinach.18) We investigated the regulatory properties of these two enzymes in a cell-free extract of obligate methylotroph OM33. a) Properties of serine-o-transacetylase. The release of free CoA shown in Fig. 3 indicates that the acylation of L-serine occurred when acetyl-coa was added as the acyl donor. On the other hand, when succinyl-coa was added instead of acetyl-coa, the release of free CoA scarcely occurred. These facts suggest the existence of serine-0-transacetylase in this bacterium. L-Methionine in-

5 L-Methionine Biosynthesis in Obligate Methylotroph 61 Table IV. Effect of l-methionine on the Synthesis of Serine-O- TRANSACETYLASE AND OAS SULFHYDRYLASE Strains OM33 and OE 120 were grown for 40hr in the presence of L-methionine as indicated. The activities of the enzymes were determined as described in Materials and Methods. I15I 7 Q. /à" / 10- / / E /y ^^ >05-1/'^r / Serine(mM) Fig. 4. Lineweaver-Burk Plot of Serine-O-transacetylase in the Presence or Absence of L-Methionine. The activity was assayed in the presence of 300 fig protein by the method described in Materials and Methods except that 10mM (3) or 20him (#) L-methionine was added or not added (O). The concentration of acetyl-coa was0.28mm. hibited the activity competitively with respect to L-serine (Fig. 4). The Ki value for l- methionine was calculated to be 7mM.SAM also inhibited the activity weakly. However, the fact that the level of this enzyme was not reduced but increased on addition of l- methionine to the growth medium, suggests Table V. Component Dependence of the Reaction of OASSulfhydrylase The complete system contained, 50 /^mol Tris-HCl, ph 7.8, 0.1 /xmol pyridoxal 5'-phosphate (PLP), 10/xmol O- acetyl-l-serine, 1.6 fimol Na2S and enzyme (101 fig protein) in a total volume of0.5 ml. Enzymewas omitted for the blank. nmol/20 min. the absence of repression of the enzyme formation by L-methionine (Table IV). b) Properties of OASsulfhydrylase. With the cell-free extract of strain OM 33, the formation of the sulfhydryl titer which was identified as L-cysteine occurred in the presence of OAS and sulfide (Table V). L-Cysteine Was not formed on the addition of L-serine instead of OAS. Omission of pyridoxal 5'- phosphate from the reaction mixture reduced the formation of L-cysteine by 20%. These results indicate that L-cysteine formation in this bacterium is catalyzed by OAS sulfhydrylase as in other microorganisms, l- Methionine inhibited the activity of this enzyme competitively with respect to OAS(Fig. 5). The Ki value for L-methionine was approximately 8mM. SAM scarcely affected the activity. The addition of L-methionine to the growth medium did not reduce the level of this enzyme (Table IV). As shown above, though both serine-otransacetylase and OASsulfhydrylase were not repressed by the addition of Lrmethionine to

6 Cystathionine å BCT v 62 Y. Morinaga, Y. Tani and H. Yamada Asp-> AspP-»ASA- HTS C TS OASS STA Horn -»OSH -^-CySH «- OAS <-Ser / / / Thr Homocystei ne r -0JHTM -1 Met SAM /OAS(mM) Fig. 5.. Lineweaver-Burk Plot of OSA Sulfhydrylase Reaction in the Presence or Absence of L-Methionine. The activity was assayed in the presence of 100 /ig protein by the method described in Materials and Methods except that lonim O) or 20niM (#) L-methionine was added or not added (O)- The concentration of Na2Swas 3.2him. the growth medium, these two enzymes were found to be subject to the feedback inhibition by L-methionine (or SAM). These facts indicate the close relationship between l- methionine biosynthesis and L-cysteine biosynthesis in this bacterium. DISCUSSION The pathway and regulatory mechanismof L-methionine biosynthesis in obligate methylotroph strain OM33 is tentatively shown in Fig. 6. In the cell-free extract of this bacterium, the acylation of L-homoserine, the first step of L-methionine biosynthesis, was found to be catalyzed by homoserine-o-transsuccinylase (Fig. 1). The acyl donor specificity in l- homoserine acylation has been reported for various microorganisms, i.e. Gram-positive bacteria such as Bacillus spp.,19~21) Arthrobacter paraffineus,20) B. flavum,6) C. glutamicum1) and C. acetophilum22) employ acetyl-coa as the acyl donor, while Gramnegative bacteria such as E. coli,3) Aerobacter aerogenes23) and S. typhimurium3) employ succinyl-coa. Strain OM 33 belongs to the latter group. Wealso found that succinyl-coa Fig. 6. Pathway and Regulation of Methionine Biosynthesis in Obligate Methylotroph OM33. --å åº, strict feedback inhibition; ->, weak inhibition; -#, assumed repression; - >, enzyme not detected yet. Asp P, /?-aspartylphosphate; ASA, aspartate /?-semialdehyde; Horn, homoserine; OSH, O-succinylhomoserine; HTS, homoserine-0-transsuccinylase; CTS, cystathionine y-synthase; BCT, /?-cystathionase; OASS, O-acetylserine sulfhydrylase; STA, serine-o-transacetylase; HTM, homocysteine transmethylase (methionine synthetase complex). is utilized for L-homoserine acylation in several obligate methanol-utilizing bacteria having the ribulose monophosphate pathway other than strain OM33 such as M. arninofaciens and so on (unpublished data). SAM in the wild-type strain, OM 33, inhibits the activity of homoserine-o-transsuccinylase significantly, whereas the inhibition by L-methionine is weak (Table II, Fig. 2). In E. coli,8) S. typhimurium2^ and B. subtilis,19) acylation of L-homoserine is reported to be synergistically inhibited by L-methionine and SAM. However, the synergistic stimulation of inhibition in the presence of both amino acids in combination was not observed with the crude extract of OM33 cells. The ethionine resistance and L-methionine excreting ability of mutant OE 120 can be accounted for by the loss of sensitivity of homoserine-o-transsuccinylase to SAM. Probably, SAMis one of the real regulatory effectors in the regulation of L-methionine biosynthesis in this bacterium. Ethionine is supposed to be activated by conversion into a homologue of SAM, which may act as a false feedback effector and inibit the growth. It is known that ethionine is converted into a homologue of SAM in Saccharomyces cerevisiae.25 ) Repression of homoserine-o-transsuccinylase by L-methionine has been reported

in many microorganisms.3~7) However, in strain OM 33 the enzyme formation was scarcely affected by L-methionine addition to the growth medium (Table I).

7 in many microorganisms.3~7) However, in strain OM 33 the enzyme formation was scarcely affected by L-methionine addition to the growth medium (Table I). This suggests that the synthesis of this enzyme is not regulated by repression, though a further study using a methionine auxotroph is necessary. The presence offeedback inhibition by L-methionine of serine-otransacetylase and OAS sulfhydrylase, which are involved in L-cysteine biosynthesis, could indicate the functional importance of the transsulfuration pathway for L-methionine biosynthesis in this bacterium. The activities of cystathionine y-synthase and /?-cystathionase, which are involved in the transsulfuration pathway, as well as the enzyme activity of direct formation of l- homocysteine from L-homoserine or homoserine derivatives, were assayed with the cellfree extract of OM33 according to the published methods.10'26'27) However, the activities of these enzymes have not been detectable so far. Thus the pathway of L-homocysteine biosynthesis in this bacterium is not clear yet. Wealso investigated the properties of transmethylation to L-homocysteine, the final step of L-methionine biosynthesis, using an intact cell system and obtained results suggesting that the enzyme or enzymes concerned in the transmethylation are repressed by l- methionine. The detailed results will be reported elsewhere.28) In conclusion, L-Methionine Biosynthesis in Obligate Methylotroph 63 from the view point of the regulation of L-methionine biosynthesis, the feedback inhibition of homoserine-otranssuccinylase by SAMseems to be the most severe regulation in strain OM 33 because modification of the enzyme by mutation permitted the over-production of L-methionine in ethionine resistant mutant OE Homoserine-O-transsuccinylase may play a key role in regulation of L-methionine biosynthesis in this bacterium. REFERENCES 1) H. Yamada, Y. Morinaga and Y. Tani, Agric. Biol. Chem., 46, 47 (1982). 2) R. J. Rowbury, Biochem. J., 82, 42P (1961). 3) R. J. Rowbury, /. Gen. MicrobioL, 37, 171 (1964). 4) H. de Robichon-Szulmajster and H. Cherest, Biochem. Biophys. Res. Commun., 28, 256 (1967). 5) S. Nagai and M. Flavin, /. Biol. Chem., 241, 3861 (1964). 6) R. Miyajima and I. Shiio, J. Biochem., 73, 1061 (1973). 7) H. Kase and 2021 (1974). K. Nakayama, Agric. Biol. Chem., 38, 8) Li-Wen Lee, J. M. Ravel and W. Shive, J. Biol. Chem., 241, 5479 (1966). 9) E. H. Ellman, Arch. Biochem. Biophys., 82, 70 (1959). 10) S. Yamagata, J. Biochem., 70, 1035 (1971). ll) O. H. Lowry, N. J. Rosebrough,A. L. FarrandR. J. Randall, /. Biol. Chem., 193, 265 (1951). 12) E. J. Simon and D. Shemin, J. Am. Chem. Soc, 75, 2520 (1953). 13) I. B. Wilson, /. Am. Chem. Soc, 74, 3205 (1952). 14) S. Nagai and M. Flavin, "Methods in Enzymology," Vol. XVII B, ed. by S. P. Colowick and N. O. Kaplan, 423. Academic Press Inc., New York, 1971, p. 15) N. M. Kredich and G. M. Tomkins, /. Biol. Chem., 241, 4955 (1966). 16) D. Gevertz, R. Amelunxen and J. M. Akagi, /. Bacteriol, 141, 1460 (1980). 17) J. L. Wiebers and H. R. Garner, /. Biol. Chem., 242, 5644 (1967). 18) J. Giovanelli and S. H. Mudd, Biochem. Biophys. Res. Commun., 27, 150 (1967). 19) A. Brush and H. Paulus, Biochem. Biophys. Res. Commun., 45, 735 (1971). 20) H. Kase and K. Nakayama, Agric. Biol. Chem., 39, 687 (1975). 21) A. Wyman and H. Paulus, /. Biol. Chem., 250, 3897 (1975). 22) Y. Murooka, K. Seto, K. Kakihara and T. Harada, Agric. Biol. Chem., 43, 1959 (1979). 23) H. Kase, K. Nakayama and S. Kinoshita, Agric. Biol. Chem., 34, 274 (1970). 24) M. A. Savin, M. Flavin and L. Slaughter, /. Bacteriol., Ill, 547 (1972). 25) F. Colombani, H. Cherest and H. de Robichon- Szulmajster, /. Bacteriol., 122, 375 (1975). 26) M. Kaplan and S. Guggenheim, "Methods in Enzymology," N. O. Kaplan, Vol. XII B, ed. Academic Press by S. Inc., P. Colowick and New York, 1971, p ) S. Guggenheim, "Methods in Enzymology," Vol. XII B, ed. by SP. P. Colowick and N. O. Kaplan. Academic Press Inc., New York, 1971, p ) Y. Morinaga, Y. Tani and H. Yamada, in preparation.

EFFECTS OF MACRO-MINERAL ELEMENTS ON GROWTH AND L-GLUTAMIC ACID FERMENTATION BY A MUTANT MICROCOCCUS GLUTAMICUS AB 100

Research Article EFFECTS OF MACRO-MINERAL ELEMENTS ON GROWTH AND L-GLUTAMIC ACID FERMENTATION BY A MUTANT MICROCOCCUS GLUTAMICUS AB 100 S. Ganguly* and A. K. Banik Dept of Chemical Engineering, Biochemical