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Citramalate formation in propionate-adapted Escherichia coli
600
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
NOTE
PN 2 1 0 7 4
Citramalate formation in propionate-adapted Escherichia coli We have recently found that cell-free extracts obtained from Escherichia coli, grown aerobically in a simple mineral-salts medium with propionate as the sole carbon source, contain enzymes which catalyze the propionyl-CoA-dependent formation of citramalate (a-methyl malate) from glyoxylate. This observation m a y be particularly significant since we have reported z earlier that these same extracts are able to catalyze the formation of a-hydroxyglutarate from glyoxylate and propionyl-CoA. The fact that citramalate is formed in these incubation mixtures suggests that it m a y be formed via an isomerization of the a-hydroxyglutarate. This type of isomerization would be completely analogous to the glutamate -+ fl-methyl aspartate 2 and methyl malonate succinatO isomerizations. The complete reaction mixture contained: 500/~moles Tris-HC1 buffer (pH 8.5), 2/~moles GSH, IO/,moles MgC12, 5 Izmoles propionyl-CoA, 2/~moles sodium [2-14C] glyoxylate containing 9.1/~C and crude cell-free extract containing 15 mg protein. Two additional tubes, one minus propionyl-CoA and one containing boiled extract were employed as controls. Incubation was anaerobic (under N2) at 37 ° for 5 h. The reaction was terminated by the addition of o.I ml of 50 % H,S04 and protein removed by centrifugation. The supernatant fluid was neutralized with K O H and placed on a column (20 m m x 200 mm) of Dowex-I (C1-). The column was washed with 200 ml of water and the citramalate eluted with a gradient of NaC1. The gradient was obtained by allowing 240 ml of I.O M NaC1 to drop, with constant stirring, into a vessel containing 240 ml of water. The flow rate was 20-24 ml/h and 4.o ml fractions were collected. As can be seen in Fig. I the peak, subsequently identified as citramalate, 5000,
o
400C 900C
io
'~200C / lOOC "" ~o~2
' s'~ ' ~
' ~'8 ' ,~o ' ~
Tubenumber
....
Fig. I. Isolation of citramalate using D o w e x - I (C1-) column. Complete enzymic reaction m i x t u r e s a n d details of column c h r o m a t o g r a p h y are described in text.
Biochim. Biophys. Acta, 97 (I965) 6oo-6o2
601
PRELIMINARY NOTES
is clearly resolved b y this procedure. There was no c i t r a m a l a t e f o r m e d in either of t h e control i n c u b a t i o n mixtures. T h e t u b e s c o n t a i n i n g this p e a k of r a d i o a c t i v i t y (Tubes 35-37) were combined, acidified to p H 2 with H2SO 4 a n d c o n t i n u o u s l y ext r a c t e d w i t h e t h e r for 24 h. F i v e t e n t h s ml w a t e r was t h e n a d d e d to the e t h e r e x t r a c t , t h e e t h e r e v a p o r a t e d u n d e r a s t r e a m of N 2 a n d aliquots of t h e aqueous solution coc h r o m a t o g r a p h e d w i t h a u t h e n t i c c i t r a m a l a t e . The results are shown in Table I. I n all solvent s y s t e m s used, only one r a d i o a c t i v e area could be found on the p a p e r s a n d in all instances e x h i b i t e d an RF identical to a u t h e n t i c c i t r a m a l a t e . R a d i o a c t i v e areas on the c h r o m a t o g r a m s were l o c a t e d b y s t r i p p i n g the p a p e r s into I - c m segments from origin to solvent front a n d counting t h e m in a liquid scintillation counter. I n order to f u r t h e r i d e n t i f y the c o m p o u n d as c i t r a m a l a t e , an aliquot of t h e aqueous e t h e r e x t r a c t was n e u t r a l i z e d with K O H a n d d r i e d at lO5 ° for I h. The salt was t h e n cooled to o ° a n d d e g r a d e d w i t h H2SO 4 according to the m e t h o d described b y STRASSMAN AND CECI 3. The m i x t u r e was t h e n distilled at IOO ° a n d the distillate, c o n t a i n i n g the acetone p r o d u c e d as the p r o d u c t of the d e g r a d a t i o n , collected in a t u b e TABLE I PAPER CHROMATOGRAPHIC IDENTIFICATION OF CITRAMALIC ACID C h r o m a t o g r a p h y was ascending at 23 ° . Spots were located after autoclaving b y s p r a y i n g w i t h 0.04 % b r o m cresol green in 95 % ethanol.
Solvent system
RF Authentic citramalic acid
E t h e r - b e n z e n e - f o r m i c a c i d - w a t e r (21 : 9 : 7 : 2, v/v) E t h e r - f o r m i c a c i d - w a t e r (5 : 2 : i, v/v) I s o a m y l f o r m a t e - f o r m i c a c i d - w a t e r (I 1:2 : i, v/v) E t h y l a c e t a t e - f o r m i c a c i d - w a t e r (io : 2 : 3, v/v) E t h y l a c e t a t e - a c e t i c a c i d - w a t e r (4 : i : 5, v/v) n - B u t a n o l - f o r m i c a c i d - w a t e r (4: 1:5, v/v) n - B u t a n o l - a c e t i c a c i d - w a t e r (4: I: I, v/v)
0.50 0.72 o.18 0.72 0.54 o.68 0.66
Enzymic product o.51
0.72 o.18 o.71 o.54 0.67 0.66
TABLE II PAPER CHROMATOGRAPHIC
IDENTIFICATION OF ACETONE 2,4-DINITROPHENYLHYDRAZONE
C h r o m a t o g r a p h y was ascending at 23 °. Spots were located with ultraviolet lamp. The b.p. range of the p e t r o l e u m ether used was 3o°-6o °.
Solvent system
RF Authentic Degradatiort acetone hydrazone product
E t h e r - p e t r o l e u m ether (5 : 95, v/v) 95 % E t h a n o l - p e t r o l e u m ether (8 : 2, v/v) n - B u t a n o l - o . 5 M N H 4 O H (I : I, v/v) M e t h a n o l - h e p t a n e (i : i, v / v ; lower phase) Petroleum e t h e r - t o l u e n e - a c e t i c a c i d - w a t e r (io: IO: 17 : 3, v/v)
o.95 o.84 o.61 o.93 o.76 0.55 I.OO
o.95 o.83 o.61 o.93 o.76 0.54 I.OO
Biochim. Biophys. Acta, 97 (1965) 60o-6o2.
602
PRELIMINARY NOTE
containing 2 ml of saturated 2,4-dinitrophenylhydrazine in 2 N HC1. After 30 min incubation at 37 °, the solution was extracted with i.o ml of ethyl acetate and aliquots chromatographed adjacent to authentic acetone 2,4-dinitrophenylhydrazone. As can be seen in Table II, the hydrazone formed as a result of the degradation migrated to the same RE as authentic acetone hydrazone. Further, the only radioactivity on the chromatograms was located at this RE. Citramalic acid has been reported to occur as an intermediate in several metabolic systems. ARNON et al) reported the formation of citramalate and itaconate during the photosynthetic assimilation of acetate by Chromatium. ADLER et al. 5 and \.VANG et al. 6 have demonstrated that in liver mitochondria pyruvate and acetyl-CoA are formed from itaconate via itaconyl-CoA and citramalyl-CoA. COOPER AND KORNBERG7 have also recently reported that in a Pseudomonas itaconate is metabolized via citramalate. BRIGHTMANAND MARTINs have reported the conversion of itaconate to succinate via citramalate. In addition, the synthesis and degradation of glutamic acid in Clostridium tetanamorphum has been shown by MUNCH-PETERSEN AND BARKER 9 to Occur via citramalate and mesaeonate. The enzymic conversion of a-hydroxyglutarate to citramalate is currently being investigated in this laboratory. The authors gratefully acknowledge the technical assistance of Miss C. URBANO. This work was supported by grants from the National Science Foundation ( G B 2020) and the National Institute of Health (AI o3866). The senior author is a National Institutes of Health Research Career Development Awardee (5-K3-AI-6928).
Albert Einstein Medical Center, Research Laboratories, Department of Biochemist~,, Philadelphia, Pa. (U.S.A.) I 2 3 4 5 6 7 8 9
H E N R Y C. REEVES SAMUEL J . A j L
H. C. REEVES AND S. J. AJL, J. Bacteriol., 84 (1962) 186. H. EGGERER, P. OVERATH, 1~. LYNEN AND E. R. STADTMAN, J. Am. Chem. Soc., 82 (196o) 2643. M. STRASSMAN AND L. N. CECI, J. Biol. Chem., 238 (1963) 2445. D. I. ARNON, M. LOSADA, i . V. TREBST AND S. OGATA, Federation Proc., 19 (196o) 328. J. ADLER, S. F. WANG AND ]-[. i . LARDY, J. Biol. Chem., 229 (1957) 865. S. F. WANG, J. ADLER AND H. A. LARDY, J. Biol. Chem., 236 (1961) 26. i{. A. COOPER AND n . L. KORNBERG, Biochem. J., 91 (1964) 82. V. BRIGHTMAN AND \V. R. MARTIN, J. Baeteriol., 82 (1961) 376. A. MUNCH-PETERSEN AND H. A. BARKER, J. Biol. Chem., 230 (1958) 649.
Received December I6th, 1964 Biochim. Biophys. Acta, 97 (1965) 600 -602
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY
NOTE
PN 2 1 0 7 4
Citramalate formation in propionate-adapted Escherichia coli We have recently found that cell-free extracts obtained from Escherichia coli, grown aerobically in a simple mineral-salts medium with propionate as the sole carbon source, contain enzymes which catalyze the propionyl-CoA-dependent formation of citramalate (a-methyl malate) from glyoxylate. This observation m a y be particularly significant since we have reported z earlier that these same extracts are able to catalyze the formation of a-hydroxyglutarate from glyoxylate and propionyl-CoA. The fact that citramalate is formed in these incubation mixtures suggests that it m a y be formed via an isomerization of the a-hydroxyglutarate. This type of isomerization would be completely analogous to the glutamate -+ fl-methyl aspartate 2 and methyl malonate succinatO isomerizations. The complete reaction mixture contained: 500/~moles Tris-HC1 buffer (pH 8.5), 2/~moles GSH, IO/,moles MgC12, 5 Izmoles propionyl-CoA, 2/~moles sodium [2-14C] glyoxylate containing 9.1/~C and crude cell-free extract containing 15 mg protein. Two additional tubes, one minus propionyl-CoA and one containing boiled extract were employed as controls. Incubation was anaerobic (under N2) at 37 ° for 5 h. The reaction was terminated by the addition of o.I ml of 50 % H,S04 and protein removed by centrifugation. The supernatant fluid was neutralized with K O H and placed on a column (20 m m x 200 mm) of Dowex-I (C1-). The column was washed with 200 ml of water and the citramalate eluted with a gradient of NaC1. The gradient was obtained by allowing 240 ml of I.O M NaC1 to drop, with constant stirring, into a vessel containing 240 ml of water. The flow rate was 20-24 ml/h and 4.o ml fractions were collected. As can be seen in Fig. I the peak, subsequently identified as citramalate, 5000,
o
400C 900C
io
'~200C / lOOC "" ~o~2
' s'~ ' ~
' ~'8 ' ,~o ' ~
Tubenumber
....
Fig. I. Isolation of citramalate using D o w e x - I (C1-) column. Complete enzymic reaction m i x t u r e s a n d details of column c h r o m a t o g r a p h y are described in text.
Biochim. Biophys. Acta, 97 (I965) 6oo-6o2
601
PRELIMINARY NOTES
is clearly resolved b y this procedure. There was no c i t r a m a l a t e f o r m e d in either of t h e control i n c u b a t i o n mixtures. T h e t u b e s c o n t a i n i n g this p e a k of r a d i o a c t i v i t y (Tubes 35-37) were combined, acidified to p H 2 with H2SO 4 a n d c o n t i n u o u s l y ext r a c t e d w i t h e t h e r for 24 h. F i v e t e n t h s ml w a t e r was t h e n a d d e d to the e t h e r e x t r a c t , t h e e t h e r e v a p o r a t e d u n d e r a s t r e a m of N 2 a n d aliquots of t h e aqueous solution coc h r o m a t o g r a p h e d w i t h a u t h e n t i c c i t r a m a l a t e . The results are shown in Table I. I n all solvent s y s t e m s used, only one r a d i o a c t i v e area could be found on the p a p e r s a n d in all instances e x h i b i t e d an RF identical to a u t h e n t i c c i t r a m a l a t e . R a d i o a c t i v e areas on the c h r o m a t o g r a m s were l o c a t e d b y s t r i p p i n g the p a p e r s into I - c m segments from origin to solvent front a n d counting t h e m in a liquid scintillation counter. I n order to f u r t h e r i d e n t i f y the c o m p o u n d as c i t r a m a l a t e , an aliquot of t h e aqueous e t h e r e x t r a c t was n e u t r a l i z e d with K O H a n d d r i e d at lO5 ° for I h. The salt was t h e n cooled to o ° a n d d e g r a d e d w i t h H2SO 4 according to the m e t h o d described b y STRASSMAN AND CECI 3. The m i x t u r e was t h e n distilled at IOO ° a n d the distillate, c o n t a i n i n g the acetone p r o d u c e d as the p r o d u c t of the d e g r a d a t i o n , collected in a t u b e TABLE I PAPER CHROMATOGRAPHIC IDENTIFICATION OF CITRAMALIC ACID C h r o m a t o g r a p h y was ascending at 23 ° . Spots were located after autoclaving b y s p r a y i n g w i t h 0.04 % b r o m cresol green in 95 % ethanol.
Solvent system
RF Authentic citramalic acid
E t h e r - b e n z e n e - f o r m i c a c i d - w a t e r (21 : 9 : 7 : 2, v/v) E t h e r - f o r m i c a c i d - w a t e r (5 : 2 : i, v/v) I s o a m y l f o r m a t e - f o r m i c a c i d - w a t e r (I 1:2 : i, v/v) E t h y l a c e t a t e - f o r m i c a c i d - w a t e r (io : 2 : 3, v/v) E t h y l a c e t a t e - a c e t i c a c i d - w a t e r (4 : i : 5, v/v) n - B u t a n o l - f o r m i c a c i d - w a t e r (4: 1:5, v/v) n - B u t a n o l - a c e t i c a c i d - w a t e r (4: I: I, v/v)
0.50 0.72 o.18 0.72 0.54 o.68 0.66
Enzymic product o.51
0.72 o.18 o.71 o.54 0.67 0.66
TABLE II PAPER CHROMATOGRAPHIC
IDENTIFICATION OF ACETONE 2,4-DINITROPHENYLHYDRAZONE
C h r o m a t o g r a p h y was ascending at 23 °. Spots were located with ultraviolet lamp. The b.p. range of the p e t r o l e u m ether used was 3o°-6o °.
Solvent system
RF Authentic Degradatiort acetone hydrazone product
E t h e r - p e t r o l e u m ether (5 : 95, v/v) 95 % E t h a n o l - p e t r o l e u m ether (8 : 2, v/v) n - B u t a n o l - o . 5 M N H 4 O H (I : I, v/v) M e t h a n o l - h e p t a n e (i : i, v / v ; lower phase) Petroleum e t h e r - t o l u e n e - a c e t i c a c i d - w a t e r (io: IO: 17 : 3, v/v)
o.95 o.84 o.61 o.93 o.76 0.55 I.OO
o.95 o.83 o.61 o.93 o.76 0.54 I.OO
Biochim. Biophys. Acta, 97 (1965) 60o-6o2.
602
PRELIMINARY NOTE
containing 2 ml of saturated 2,4-dinitrophenylhydrazine in 2 N HC1. After 30 min incubation at 37 °, the solution was extracted with i.o ml of ethyl acetate and aliquots chromatographed adjacent to authentic acetone 2,4-dinitrophenylhydrazone. As can be seen in Table II, the hydrazone formed as a result of the degradation migrated to the same RE as authentic acetone hydrazone. Further, the only radioactivity on the chromatograms was located at this RE. Citramalic acid has been reported to occur as an intermediate in several metabolic systems. ARNON et al) reported the formation of citramalate and itaconate during the photosynthetic assimilation of acetate by Chromatium. ADLER et al. 5 and \.VANG et al. 6 have demonstrated that in liver mitochondria pyruvate and acetyl-CoA are formed from itaconate via itaconyl-CoA and citramalyl-CoA. COOPER AND KORNBERG7 have also recently reported that in a Pseudomonas itaconate is metabolized via citramalate. BRIGHTMANAND MARTINs have reported the conversion of itaconate to succinate via citramalate. In addition, the synthesis and degradation of glutamic acid in Clostridium tetanamorphum has been shown by MUNCH-PETERSEN AND BARKER 9 to Occur via citramalate and mesaeonate. The enzymic conversion of a-hydroxyglutarate to citramalate is currently being investigated in this laboratory. The authors gratefully acknowledge the technical assistance of Miss C. URBANO. This work was supported by grants from the National Science Foundation ( G B 2020) and the National Institute of Health (AI o3866). The senior author is a National Institutes of Health Research Career Development Awardee (5-K3-AI-6928).
Albert Einstein Medical Center, Research Laboratories, Department of Biochemist~,, Philadelphia, Pa. (U.S.A.) I 2 3 4 5 6 7 8 9
H E N R Y C. REEVES SAMUEL J . A j L
H. C. REEVES AND S. J. AJL, J. Bacteriol., 84 (1962) 186. H. EGGERER, P. OVERATH, 1~. LYNEN AND E. R. STADTMAN, J. Am. Chem. Soc., 82 (196o) 2643. M. STRASSMAN AND L. N. CECI, J. Biol. Chem., 238 (1963) 2445. D. I. ARNON, M. LOSADA, i . V. TREBST AND S. OGATA, Federation Proc., 19 (196o) 328. J. ADLER, S. F. WANG AND ]-[. i . LARDY, J. Biol. Chem., 229 (1957) 865. S. F. WANG, J. ADLER AND H. A. LARDY, J. Biol. Chem., 236 (1961) 26. i{. A. COOPER AND n . L. KORNBERG, Biochem. J., 91 (1964) 82. V. BRIGHTMAN AND \V. R. MARTIN, J. Baeteriol., 82 (1961) 376. A. MUNCH-PETERSEN AND H. A. BARKER, J. Biol. Chem., 230 (1958) 649.
Received December I6th, 1964 Biochim. Biophys. Acta, 97 (1965) 600 -602