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Accumulation of l -cystathionine by an Escherichia coli mutant deficient in cystathionine beta-lyase
OFBioscmucaANDBIO~G~~~ Vol.94,No.2,1X$-181.2002 Joma.
Accumulation of L-Cystathionine by an Escherichia coli Mutant Deficient in Cystathionine Beta-Lyase TAKUYA NISHI,’ KAZUHIKO TANAKA: YOSHITAKE TANAKA,3 KAZUMI ARAKI,‘* KAZUO FURIHATA,4MASAYUKI ONODERA,5 ANDKIYOSHI TODA5 Graduate School of Integrated Science and Art, University of East Asia, 2-i ~ch~nom~a-ga~enma~hi, Shimonaseki City, Yamaguchi 75f-0807, Japan*’ Faculty of Engineering, ~niversi~ of East Asia, 2-1 rchinomiya-ga~enmachi, Shimonose~ City Yama~chi 751-0807, Japan2 Research Centerfor ~iolo~.ca~s~ The Kitasuto vitiate, 6-111 Arai, ~itamoto City, ~it~a 364-~~26, Japan,’ Graduate ~~0~ of Agriculture and Life Science, Universitv of Tokyo, 1-l-I Yayoi, Bunkyo-Ku, To&o 113-0032,J~an,4~ Faculty of Engineering, Niigata Institute of Technology, 1719 Fujihashi, Kasiwazaki City Niigata 945-1195, Japan5
Received13August2001/Accepted 22May2002 An Escherichia coli mutant deficient in cystathionine j3-lyase was found to accumulate a substance detectable by ninhydrin reaction and chloride platiuic acid reaction in its cells (rarely in the culture supernatant) when cultured with a limited amount (JO-200 @ml) of L-methionine to support the growth. The product was released by freezing treatment and isolated by ion-exchange chromatography (cation exchange resin: Daiaion SKlB). It was identified as L-cystathioniue by liquid chromatography-mass spectrometry, 13C-and *H-nuclear magnetic resonance analyses and huh-buoyance liquid ch~mat~~phy (as its 2~,4,~te~a-~-ace~l-~~glucop~no~l isothiocyanate derivative). [Key words:
L-cystathionine,cystathionine 8-lyase]
L-Cystathionine is an important intermediate in sulfur amino acid metabolism in many organisms (1, 2). It occurs in larger amounts in mammalian brain than in other tissues (3). The L-cystathionine content in the brain is related to inherited metabolic diseases (4-6). Moreover, it is expected to have a prophylatic effect on drug-induced injury by acting as a prodrug for cysteine release (7). To provide a stable supply for further medical research on its prophylatic and therapeutic effects, some enzymatic methods for the preparation of L-cystathionine have been reported which use cystathionine y-lyase (EC 4.4.1.1) (8) and cystathionine y-synthase (EC 4.2.99.9) (9) from bacterial sources. L-Homoserine and O-acyl+homoserine together with L-cysteine are required as substrates in the respective reactions. To establish a more direct fermentation process for the production of L-cystathionine, we aimed at culturing an Escherichiu colt mutant, CAG18475 (MetC-) (10) (kindly supplied by Dr. M. Berlyn of Yale University), having a deficiency in cysta~ionine p-lyase (EC 4.4.1.8) which is known to metabolize cystathionine in the methionine synthetic pathway of E. co& (1). Our rationale was that the genetic block of the enzyme catalyzing the metabolism of a particular intermediate in an amino acid synthetic pathway and the deregulation of its synthetic pathway enzymes caused by a deficiency of the regulating amino acid would trigger the overproduction of the intermediate in the mutant requiring the regulator amino acid because of the genetic
block (11, 12). The fermentation for cystathionine production was carried out as follows: a loop of bacterial cells grown on a Iuria-Bertani agar slant (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 20 g of agar per liter, pH 7) was inoculated in a 1OO-mlErlemneyer flask containing 30 ml of a seed medium (10 g of D-glucose, 10 g of pol~ptone, 5g of yeast extract per liter, pH 7) and incubated for 16-l 8 h on a reciprocal shaker (model T-22$ Thomas Kagaku, Tokyo) operated at 66 rpm and 30°C. The grown cells were collected by centrifugation of the culture and suspended in one-third volume of the supernatant liquid. The cell suspension thus concentrated was inoculated with an inoculum size of 0.7 ml in a 300-ml Erlenmeyer flask containing 7 ml of a fermentation medium, and incubated on a reciprocal shaker (model AT-12; Thomas Kagaku) operated at 140 rpm and 30°C for 44 h. The basal medium composition was as follows: 60 g of fructose (added 30 g at 0 time and 30 g at cultivation time of 20 h), 10 g of @H&SO,, 0.01 g of FeSO,. 7H,O, 0,007 g of MnSO,. 4H,O, 0.5 g of MgSO,. 7H,O, 1.5 g of KH*PO.,, 0.5 g of KJ-IPO,, 0.1 g of NaCl, 1 g of yeast extract (Oriental Kobo, Tokyo), 20g of CaCO,, 0.1 g of uracil per liter and various amounts of L-methionine (pH 7.0 adjusted with NH,OH). L-Methionine and (NH,),SO,, and CaCO, were sterilized separately from the other constituents by autoclaving for 10 min, and heating at 170°C for 30 min, respectively. The fermentations were carried out by supplementing of O-500 pg/ml L-methionine to the medium several times. A substance, which was later identified as L-cystathionine as will be described below, was rarely (once per recent six fer-
* Correspondingauthor. e-mail:[email protected] fax: +81-(0)832-.57-l 166
phone: +81-(0)832-574089
178
VOL.94,2002
NOTES
0
200 300 400 L-Methionine added ( nLg/ml)
100
179
500
FIG. 1. Intracellular accumulation of L-cystathionine by an E. co/i MetC- mutant. Fermentation was carried out at the given concentrations of L-methionine and each culture obtained was divided into two parts. After treatment with (circle) or without (triangle) freezing-thawing and centrifugation thereof, the supematant fractions obtained were assayed for L-cystathionine content by high-performance liquid chromatography as described in the legend for Fig. 4. Bacterial growth (square) was measured by measuring the absorbance at 660 nm of the culture, after dissolving CaCO, in the medium by HCl and loo-fold dilution, through a light path of 10 mm.
mentations) detected in the culture supernatant, and the accumulation was not always reproduced. It was as certained later that in most cases, the substance accumulated within the cells cultured with limiting amounts of 50-200 pg/ml Lmethionine to support the growth, as exemplified in Fig. 1. It was easily released from the cells by thawing after freezing at -20°C. The product was detectable by ninhydrin reaction and chloride platinic acid reaction, and its migration coincided with that of authentic cystathionine in paper chromatography with n-butanol-acetic acid-water (5 : 2 :2), n-propanolwater (7 : 3) and acetone-n-butanol-water-diethylamine (5 : 5 : 2 : 1) as solvents, In the amino acid analysis (model JLC-300; Nihon Denshi, Tokyo) of a sample prepared by centrifugation after the freezing treatment of the whole culture obtained by fermentation with 125 pg/ml L-methionine, the highest peak had a retention time that coincided with that of cystathionine (Fig. 2). Two other peaks (Byp 1 and Byp 2 in Fig. 2), the second and third highest peaks, the retention times of which coincided with those of O-acetylserine and O-succinylhomoserine, respectively, in other runs (data not shown) and the fourth highest peak corresponding to isoleucine were observed. A wild-type E. coli K-12 strain, EMG2 (kindly supplied by Dr. Bachmann of Yale University), did not accumulate any of these substances in significant amounts (data not shown). To isolate the substance corresponding to cystathionine, 160 ml of the supernatant solution prepared after the freezing treatment as described above was fractionated using a column (2.6 cm diameter and 30 cm height) containing a cation-exchange resin, Daiaion SKlB (H’ type; Nihon Rensui, Fukuoka), after diluting twice with water and adjusting the pH to 3 with aqueous HCl. After washing three times with the void volume of water, the column was eluted with the same volume of 0.5 N NH,OH and 2 N NH,OH at a rate of 160 ml per h. 160-ml fractions were collected, and then the presence of cystathionine in the third fraction eluted with 0.5 N NH,OH and all the three fractions eluted
Retention time (min) FIG. 2. Amino acid analysis of the culture of an E. co/i MetC- mutant. A culture obtained by fermentation with 125 ug of L-methionine per ml was subjected to freezing treatment, and the supematant obtained after centrifugation was analyzed. ABA, Aminobutyric acid; Cyst., L-cystathionine; Byp 1 and Byp 2, peaks of by-products corresponding to O-acetylserine and O-succinylhomoserine, respectively.
with 2 N NH,OH was confirmed by thin-layer chromatography. These fractions were collected and concentrated in vucuo to one-tenth of the original volume and cooled in a refrigerator after filtration. The precipitate that appeared was washed with 50% methanol, isolated and used in further analyses. The electrospray ionization-mass spectrometry (MS) spectrum of the isolated sample in the liquid chromatography-MS analysis showed a molecular ion at m/z 223 (M+H)‘, which coincided with that of cystathionine. The ‘H- and r3C-nuclear magnetic resonance (NMR) spectroscopy data and the structure of cystathionine are shown in Table 1 and Fig. 3, respectively. Seven signals observed in the 13C-NMR spectrum were proved to be due to three methylene carbons, two methine carbons and two quaternary carbons. Relationships between proton and carbon signals were established by the field-gradient heteronuclear single quantum coherence method. The connectivities between carbons in the molecule were proved indirectly by the use of the field-gradient heteronuclear multiple bond correlation method. In addition, the ‘H- and 13C-NMR chemical shifts of the sample were in complete agreement with those of an authentic sample of L-cystathionine (product of Sigma-Aldrich/Japan, Tokyo, indicated purity of approximately 90%). To determine the configuration of cystathionine, the sample was analyzed by high-performance liquid chromatography according to the method of Takahashi et al. (13), which successfully resolved the stereoisomers of 2,6diaminopimelic acid, with some modifications. The chromatographic system consisted of a high-pressure pump equipped with a universal valve injector (Shimadzu, Kyoto), a Develosil ODS-5 column (100x6.0 mm i.d., particle size 5 urn; Nomura Chemical, Seto) and an SPD-1OA spectrophotometric detector (Shimadzu). As shown in Fig. 4A,
180
NISHI ET AL.
J. BIOSCI.BIOENG., TABLE 1. ‘H- and “C-NMR data for cystathionine in D,O 13C(mult)
No. 1 2 3 4 1’ 2’ 3’
‘H (multi’ J [Hzl)
172.1 (s) 52.2 (d) 30.1 (t) 27.7 (t) 171.1 (s) 52.9 (d) 31.6 (t)
HMBC (‘H) correlation 2.24,2.34,4.26 2.24,2.34,2.81,2.84 2.81,2.84,4.26 2.24,2.34,2.81,2.84, 3.18,3.28,4.38 3.18, 3.28 2.81,2.84,4.38
4.26 (t 7.0,7.0) 2.24 (m), 2.34 (m) 2.81 (m), 2.84 (m) 4.38 (dd 7.1,4.4) 3.18 (dd 15.0, 7.1) 3.28 (dd 15.0,4.4)
3.18, 3.28
* C-H multiplicities: s, singlet; d, doublet; t, triplet. b H-H multiplicities: t, triplet; m, multiplet; dd, double-doublet. NMR spectra were recorded on a JNM-A-500 (500 MHz) spectrometer (JEOL, Tokyo). A 5 mg of sample cystathionine was dissolved in 0.2 ml of D,0+0.02 ml of 20% DCL and a micro-sample tube was used. The methyl groups of DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) were used as the internal standard and referred to as 0 ppm in the ‘H-NMR chemical shifts. Dioxane appearing at 67.4 ppm was used as reference for the r3C-NMR chemical shifts.
NH2
HOOd-&
-
:H2 4H2
-
s -&2-&i-iO0t.l
I
thH2
FIG 3. Structure of cystathionine.
the 2,3,4,6-tetra-O-acetyl-/!I-D-glucopyranosyl isothiocyanate (GITC) derivative of authentic chemically synthesized cystathionine (product of Sigma-Aldrich/Japan, indicated purity, minimum of 90%) gave four main peaks designated by a, b, c and d. The areas calculated conveniently from the product of the height and the half-width of the four peaks were almost equal. Accordingly, these must be due to the four isomers (L-, L-&o-, D- and D-allo-forms) of cystathionine. Both authentic L-cystathionine (Fig. 4B) and the examined sample (Fig. 4C) gave a main peak at the same
position as peak a of the synthesized sample (Fig. 4A). These results show that the examined product is the L-form of cystathionine. It has been known that L-cystathionine is an intermediate of methionine synthesis and its synthesis is subject to the feedback regulation by L-methionine and S-adenosyl+ metbionine (1). The accumulation of L-cystathionine in the mutant under the L-metbionine limited condition studied here may be caused by the blockage of the metabolism of L-cystathionine and the deregulation on the biosynthetic enzymes as a result of the defect in cystathionine P-lyase and hence L-methionine deficiency. It is known in E. coli that L-isoleucine and L-methionine are synthesized via L-homoserine as a common biosynthetic precursor and, O-acetyl+serine and 0-succinyl+homoserine are the precursors for the synthesis of L-cystathionine. The accumulation of L-isoleucine and these acylated amino acids as by-products, although their identification needs to be confirmed, may be explained
A
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
25
Retention time @in) FIG 4. High-performance liquid chromatograms of GITC derivatives of cystathionine stereoisomers and test sample. (A) Authentic chemically synthesized cystathionine; (B) authentic L-cystathionine; (C) test sample isolated from the culture of E. coli mutant CAG18475 (MetC-). Forty microliters of the sample solution (5 mg/ml) was used for the derivation of GITC without esteritication pretreatment, and 12 ul was injected to the column. Mobile phase, Methanol45 mM ammonium phosphate (pH 6.5)=40: 60; flow rate, 0.5 ml/min; detection, UV absorbance at 250 mn; temperature, 45°C.
VOL. 94,2002
NOTES
in relation to the regulation by L-methionine and its derivatives as described above. We thank the Institute of Technology of Kyowa Hal&o Kogyo Co., Ltd. for the amino acid analysis and The E. coli Genetic Stock Center of Yale University for the supply of E. coli strains. We also thank Mr. Eiichiro Yamada and Mr. Toshio Oyoshi for their supPort in the experiment.
REFERENCES 1. Rowbury, R. J.: Methionine biosynthesis and its regulation, p. 191-211. In Herrmann, K. M. and Somerville, R. L. (ed.), Amino acids: biosynthesis and genetic regulation. AddisonWesley Publishing, London, Amsterdam, Don Mills, Ontario, Sydney, Tokyo (1983). 2. MacCoss, M. J., Fukagawa, N. K., and Mattews, D. E.: Measurement of intracellular sulfur amino acid metabolism in humans. Am. J. Physiol. Endocrinol. Metab., 280, E947E955 (2001). 3. Tallan, H. H., Moore. S., and Stin, W. H.: L-Cystathionine in human brain. J. Biol. Chem., 230,707-716 (1958). 4. Gerritson, T. and Waisman, H.A.: Homocysteinuria: absence of cystathionine in the brain. Science, 145, 588 (1964). 5. Okumura, N., Otsuki, S., and Kameyama, A.: Studies on free amino acids in human brain. J. Biochem., 47, 315-320 (1960). 6. Haraguchi, H.: Inherited metabolic disorders of the transsulfuration pathway. Nihon Rinsho, 50, 108-l 14 (1992).
181
7. Kitamura, Y., Kamisaki, Y., and Itoh, T.: Hepatoprotective effects of cystathionine against acetaminophen-induced necrosis. J. Pharmacol. Exp. Ther., 250, 667-671 (1989). 8. Yamada, H., Kanzaki, H., and Nagasawa, T.: Synthesis of L-cystathionine by the y-replacement reaction of cystathionine y-lyase from Streptomycesphaeochromogertes. J. Biotechnol., 1,205-217 (1984). 9. Kanzaki, H., Kobayashi, M., Nagasawa, T., and Yamada, H.: Production of L-cystathionine using bacterial cystathionine y-synthase. Appl. Microbial. Biotechnol., 25, 322-326 (1987). 10. Singer, M., Baker, T.A., Schnitzler, G., Deischel, S.M., Gael, M., Dove, W., Jaacks, K. J., Grossman, A. D., and Erickson, J. W.: A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbial. Rev., 53, l-24 (1989). 11. Araki, K. and Oseki, T.: Amino acids: survey, p. 504-57 1. In Kroschwitz, J. I. and Grant, M. H. (ed.), Kirk-Gthmer encyclopedia of chemical technology, 4th ed., vol. 2. John Wiley & Sons, New York (1992). 12. Nakayama, K.: Amino acids, p. 748-801. In Read, G. (ed.), Prescott and Dunn’s industrial microbiology, 4th ed. Avi Publishing, Westport, Connecticut (1982). 13. Takahashi, Y., Iwai, Y., Tomoda, H., Nimura, N., Kinoshita, T., and Omura, S.: Optical resolution of 2,6-diaminopimelic acid stereoisomers by high performance liquid chromatography for the chemotaxonomy of actinomycete strains. J. Gen. Appl. Microbial., 35,27-32 (1989).
Accumulation of L-Cystathionine by an Escherichia coli Mutant Deficient in Cystathionine Beta-Lyase TAKUYA NISHI,’ KAZUHIKO TANAKA: YOSHITAKE TANAKA,3 KAZUMI ARAKI,‘* KAZUO FURIHATA,4MASAYUKI ONODERA,5 ANDKIYOSHI TODA5 Graduate School of Integrated Science and Art, University of East Asia, 2-i ~ch~nom~a-ga~enma~hi, Shimonaseki City, Yamaguchi 75f-0807, Japan*’ Faculty of Engineering, ~niversi~ of East Asia, 2-1 rchinomiya-ga~enmachi, Shimonose~ City Yama~chi 751-0807, Japan2 Research Centerfor ~iolo~.ca~s~ The Kitasuto vitiate, 6-111 Arai, ~itamoto City, ~it~a 364-~~26, Japan,’ Graduate ~~0~ of Agriculture and Life Science, Universitv of Tokyo, 1-l-I Yayoi, Bunkyo-Ku, To&o 113-0032,J~an,4~ Faculty of Engineering, Niigata Institute of Technology, 1719 Fujihashi, Kasiwazaki City Niigata 945-1195, Japan5
Received13August2001/Accepted 22May2002 An Escherichia coli mutant deficient in cystathionine j3-lyase was found to accumulate a substance detectable by ninhydrin reaction and chloride platiuic acid reaction in its cells (rarely in the culture supernatant) when cultured with a limited amount (JO-200 @ml) of L-methionine to support the growth. The product was released by freezing treatment and isolated by ion-exchange chromatography (cation exchange resin: Daiaion SKlB). It was identified as L-cystathioniue by liquid chromatography-mass spectrometry, 13C-and *H-nuclear magnetic resonance analyses and huh-buoyance liquid ch~mat~~phy (as its 2~,4,~te~a-~-ace~l-~~glucop~no~l isothiocyanate derivative). [Key words:
L-cystathionine,cystathionine 8-lyase]
L-Cystathionine is an important intermediate in sulfur amino acid metabolism in many organisms (1, 2). It occurs in larger amounts in mammalian brain than in other tissues (3). The L-cystathionine content in the brain is related to inherited metabolic diseases (4-6). Moreover, it is expected to have a prophylatic effect on drug-induced injury by acting as a prodrug for cysteine release (7). To provide a stable supply for further medical research on its prophylatic and therapeutic effects, some enzymatic methods for the preparation of L-cystathionine have been reported which use cystathionine y-lyase (EC 4.4.1.1) (8) and cystathionine y-synthase (EC 4.2.99.9) (9) from bacterial sources. L-Homoserine and O-acyl+homoserine together with L-cysteine are required as substrates in the respective reactions. To establish a more direct fermentation process for the production of L-cystathionine, we aimed at culturing an Escherichiu colt mutant, CAG18475 (MetC-) (10) (kindly supplied by Dr. M. Berlyn of Yale University), having a deficiency in cysta~ionine p-lyase (EC 4.4.1.8) which is known to metabolize cystathionine in the methionine synthetic pathway of E. co& (1). Our rationale was that the genetic block of the enzyme catalyzing the metabolism of a particular intermediate in an amino acid synthetic pathway and the deregulation of its synthetic pathway enzymes caused by a deficiency of the regulating amino acid would trigger the overproduction of the intermediate in the mutant requiring the regulator amino acid because of the genetic
block (11, 12). The fermentation for cystathionine production was carried out as follows: a loop of bacterial cells grown on a Iuria-Bertani agar slant (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 20 g of agar per liter, pH 7) was inoculated in a 1OO-mlErlemneyer flask containing 30 ml of a seed medium (10 g of D-glucose, 10 g of pol~ptone, 5g of yeast extract per liter, pH 7) and incubated for 16-l 8 h on a reciprocal shaker (model T-22$ Thomas Kagaku, Tokyo) operated at 66 rpm and 30°C. The grown cells were collected by centrifugation of the culture and suspended in one-third volume of the supernatant liquid. The cell suspension thus concentrated was inoculated with an inoculum size of 0.7 ml in a 300-ml Erlenmeyer flask containing 7 ml of a fermentation medium, and incubated on a reciprocal shaker (model AT-12; Thomas Kagaku) operated at 140 rpm and 30°C for 44 h. The basal medium composition was as follows: 60 g of fructose (added 30 g at 0 time and 30 g at cultivation time of 20 h), 10 g of @H&SO,, 0.01 g of FeSO,. 7H,O, 0,007 g of MnSO,. 4H,O, 0.5 g of MgSO,. 7H,O, 1.5 g of KH*PO.,, 0.5 g of KJ-IPO,, 0.1 g of NaCl, 1 g of yeast extract (Oriental Kobo, Tokyo), 20g of CaCO,, 0.1 g of uracil per liter and various amounts of L-methionine (pH 7.0 adjusted with NH,OH). L-Methionine and (NH,),SO,, and CaCO, were sterilized separately from the other constituents by autoclaving for 10 min, and heating at 170°C for 30 min, respectively. The fermentations were carried out by supplementing of O-500 pg/ml L-methionine to the medium several times. A substance, which was later identified as L-cystathionine as will be described below, was rarely (once per recent six fer-
* Correspondingauthor. e-mail:[email protected] fax: +81-(0)832-.57-l 166
phone: +81-(0)832-574089
178
VOL.94,2002
NOTES
0
200 300 400 L-Methionine added ( nLg/ml)
100
179
500
FIG. 1. Intracellular accumulation of L-cystathionine by an E. co/i MetC- mutant. Fermentation was carried out at the given concentrations of L-methionine and each culture obtained was divided into two parts. After treatment with (circle) or without (triangle) freezing-thawing and centrifugation thereof, the supematant fractions obtained were assayed for L-cystathionine content by high-performance liquid chromatography as described in the legend for Fig. 4. Bacterial growth (square) was measured by measuring the absorbance at 660 nm of the culture, after dissolving CaCO, in the medium by HCl and loo-fold dilution, through a light path of 10 mm.
mentations) detected in the culture supernatant, and the accumulation was not always reproduced. It was as certained later that in most cases, the substance accumulated within the cells cultured with limiting amounts of 50-200 pg/ml Lmethionine to support the growth, as exemplified in Fig. 1. It was easily released from the cells by thawing after freezing at -20°C. The product was detectable by ninhydrin reaction and chloride platinic acid reaction, and its migration coincided with that of authentic cystathionine in paper chromatography with n-butanol-acetic acid-water (5 : 2 :2), n-propanolwater (7 : 3) and acetone-n-butanol-water-diethylamine (5 : 5 : 2 : 1) as solvents, In the amino acid analysis (model JLC-300; Nihon Denshi, Tokyo) of a sample prepared by centrifugation after the freezing treatment of the whole culture obtained by fermentation with 125 pg/ml L-methionine, the highest peak had a retention time that coincided with that of cystathionine (Fig. 2). Two other peaks (Byp 1 and Byp 2 in Fig. 2), the second and third highest peaks, the retention times of which coincided with those of O-acetylserine and O-succinylhomoserine, respectively, in other runs (data not shown) and the fourth highest peak corresponding to isoleucine were observed. A wild-type E. coli K-12 strain, EMG2 (kindly supplied by Dr. Bachmann of Yale University), did not accumulate any of these substances in significant amounts (data not shown). To isolate the substance corresponding to cystathionine, 160 ml of the supernatant solution prepared after the freezing treatment as described above was fractionated using a column (2.6 cm diameter and 30 cm height) containing a cation-exchange resin, Daiaion SKlB (H’ type; Nihon Rensui, Fukuoka), after diluting twice with water and adjusting the pH to 3 with aqueous HCl. After washing three times with the void volume of water, the column was eluted with the same volume of 0.5 N NH,OH and 2 N NH,OH at a rate of 160 ml per h. 160-ml fractions were collected, and then the presence of cystathionine in the third fraction eluted with 0.5 N NH,OH and all the three fractions eluted
Retention time (min) FIG. 2. Amino acid analysis of the culture of an E. co/i MetC- mutant. A culture obtained by fermentation with 125 ug of L-methionine per ml was subjected to freezing treatment, and the supematant obtained after centrifugation was analyzed. ABA, Aminobutyric acid; Cyst., L-cystathionine; Byp 1 and Byp 2, peaks of by-products corresponding to O-acetylserine and O-succinylhomoserine, respectively.
with 2 N NH,OH was confirmed by thin-layer chromatography. These fractions were collected and concentrated in vucuo to one-tenth of the original volume and cooled in a refrigerator after filtration. The precipitate that appeared was washed with 50% methanol, isolated and used in further analyses. The electrospray ionization-mass spectrometry (MS) spectrum of the isolated sample in the liquid chromatography-MS analysis showed a molecular ion at m/z 223 (M+H)‘, which coincided with that of cystathionine. The ‘H- and r3C-nuclear magnetic resonance (NMR) spectroscopy data and the structure of cystathionine are shown in Table 1 and Fig. 3, respectively. Seven signals observed in the 13C-NMR spectrum were proved to be due to three methylene carbons, two methine carbons and two quaternary carbons. Relationships between proton and carbon signals were established by the field-gradient heteronuclear single quantum coherence method. The connectivities between carbons in the molecule were proved indirectly by the use of the field-gradient heteronuclear multiple bond correlation method. In addition, the ‘H- and 13C-NMR chemical shifts of the sample were in complete agreement with those of an authentic sample of L-cystathionine (product of Sigma-Aldrich/Japan, Tokyo, indicated purity of approximately 90%). To determine the configuration of cystathionine, the sample was analyzed by high-performance liquid chromatography according to the method of Takahashi et al. (13), which successfully resolved the stereoisomers of 2,6diaminopimelic acid, with some modifications. The chromatographic system consisted of a high-pressure pump equipped with a universal valve injector (Shimadzu, Kyoto), a Develosil ODS-5 column (100x6.0 mm i.d., particle size 5 urn; Nomura Chemical, Seto) and an SPD-1OA spectrophotometric detector (Shimadzu). As shown in Fig. 4A,
180
NISHI ET AL.
J. BIOSCI.BIOENG., TABLE 1. ‘H- and “C-NMR data for cystathionine in D,O 13C(mult)
No. 1 2 3 4 1’ 2’ 3’
‘H (multi’ J [Hzl)
172.1 (s) 52.2 (d) 30.1 (t) 27.7 (t) 171.1 (s) 52.9 (d) 31.6 (t)
HMBC (‘H) correlation 2.24,2.34,4.26 2.24,2.34,2.81,2.84 2.81,2.84,4.26 2.24,2.34,2.81,2.84, 3.18,3.28,4.38 3.18, 3.28 2.81,2.84,4.38
4.26 (t 7.0,7.0) 2.24 (m), 2.34 (m) 2.81 (m), 2.84 (m) 4.38 (dd 7.1,4.4) 3.18 (dd 15.0, 7.1) 3.28 (dd 15.0,4.4)
3.18, 3.28
* C-H multiplicities: s, singlet; d, doublet; t, triplet. b H-H multiplicities: t, triplet; m, multiplet; dd, double-doublet. NMR spectra were recorded on a JNM-A-500 (500 MHz) spectrometer (JEOL, Tokyo). A 5 mg of sample cystathionine was dissolved in 0.2 ml of D,0+0.02 ml of 20% DCL and a micro-sample tube was used. The methyl groups of DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) were used as the internal standard and referred to as 0 ppm in the ‘H-NMR chemical shifts. Dioxane appearing at 67.4 ppm was used as reference for the r3C-NMR chemical shifts.
NH2
HOOd-&
-
:H2 4H2
-
s -&2-&i-iO0t.l
I
thH2
FIG 3. Structure of cystathionine.
the 2,3,4,6-tetra-O-acetyl-/!I-D-glucopyranosyl isothiocyanate (GITC) derivative of authentic chemically synthesized cystathionine (product of Sigma-Aldrich/Japan, indicated purity, minimum of 90%) gave four main peaks designated by a, b, c and d. The areas calculated conveniently from the product of the height and the half-width of the four peaks were almost equal. Accordingly, these must be due to the four isomers (L-, L-&o-, D- and D-allo-forms) of cystathionine. Both authentic L-cystathionine (Fig. 4B) and the examined sample (Fig. 4C) gave a main peak at the same
position as peak a of the synthesized sample (Fig. 4A). These results show that the examined product is the L-form of cystathionine. It has been known that L-cystathionine is an intermediate of methionine synthesis and its synthesis is subject to the feedback regulation by L-methionine and S-adenosyl+ metbionine (1). The accumulation of L-cystathionine in the mutant under the L-metbionine limited condition studied here may be caused by the blockage of the metabolism of L-cystathionine and the deregulation on the biosynthetic enzymes as a result of the defect in cystathionine P-lyase and hence L-methionine deficiency. It is known in E. coli that L-isoleucine and L-methionine are synthesized via L-homoserine as a common biosynthetic precursor and, O-acetyl+serine and 0-succinyl+homoserine are the precursors for the synthesis of L-cystathionine. The accumulation of L-isoleucine and these acylated amino acids as by-products, although their identification needs to be confirmed, may be explained
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Retention time @in) FIG 4. High-performance liquid chromatograms of GITC derivatives of cystathionine stereoisomers and test sample. (A) Authentic chemically synthesized cystathionine; (B) authentic L-cystathionine; (C) test sample isolated from the culture of E. coli mutant CAG18475 (MetC-). Forty microliters of the sample solution (5 mg/ml) was used for the derivation of GITC without esteritication pretreatment, and 12 ul was injected to the column. Mobile phase, Methanol45 mM ammonium phosphate (pH 6.5)=40: 60; flow rate, 0.5 ml/min; detection, UV absorbance at 250 mn; temperature, 45°C.
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NOTES
in relation to the regulation by L-methionine and its derivatives as described above. We thank the Institute of Technology of Kyowa Hal&o Kogyo Co., Ltd. for the amino acid analysis and The E. coli Genetic Stock Center of Yale University for the supply of E. coli strains. We also thank Mr. Eiichiro Yamada and Mr. Toshio Oyoshi for their supPort in the experiment.
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