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Biosynthesis of quinolinic acid in a cell-free system
199
SHORT COMMUNICATIONS
boxylasen, TM when 14C0~. is introduced. However, malate remains labelled in C-4 in photosynthesising Atriplex leaves which suggests that little is formed by a second carboxylation with a labelled C 8 compound from the carbon-reduction cycle. Carbon fixed in the C-4 position of malate or aspartate cannot contribute directly to carbohydrate synthesis via known pathways. If this fixation is followed by transcarboxylation with another CO, acceptor, e.g. ribulose 1,5-diphosphate, as suggested in sugar canO, labile malate and aspartate pools in the chloroplast is may serve as a temporary store of carbon. The high affinity for CO, of such a carboxylation system may reasonably explain photosynthetic carbon fixation at physiological CO s concentrations.
Botany Department, University of Adelaide, Adelaide (Australia)
C. B.
OSMOND*
I M. CALVIN AND J. A. BASSHAM, The Photosynthesis of Carbon Compounds, B e n j a m i n , N e w York, 1962. 2 H. P. KORTSCHAK, C. E. HARTT AND G. O. BURR, Plant Physiol., 4 ° (1965) 209. 3 M. D. HATCH AND C. R. SLACK, Biochem. J., IOi (1966) lO 3. 4 M. D. HATCH, C. R. SLACK AND H. S. JOHNSON, Biochem. J., lO2 (1967) 417 . 5 S. ARONOFF, Techniques of Radiobioehemistry, I o w a S t a t e U n i v e r s i t y Press, A m e s , 1956. 6 P. M. NOSSAL, Biochem. J., 49 (1951) 4o7. 7 L. R. ROSENBERG, J. B. CAPINDALE AND F. R. WHATLEY, Nature, 18I (1958) 632. 8 C. B. OSMOND AND P. N. AVADAHNI, in p r e p a r a t i o n . 9 A. A. BENSON, S. KAWANCHI, P. HAYES AND M. CALVIN, J. Am. Chem. Sot., 74 (1952) 4477. io D. GRAHAM AND C. P. WITTINGHAM, in p r e p a r a t i o n . II D. A. WALKER, Biol. Rev., 37 (1962) 215. 12 E. RACKER, Arch. Biochem. Biophys., 69 (1957) 3oo. 13 A. OGUN AND C. R. STOCKING, Plant Physiol., 4 ° (1965) 825.
Received March 6th, 1967 * P r e s e n t a d d r e s s : B o t a n y D e p a r t m e n t , U n i v e r s i t y of C a m b r i d g e , Cambridge, E n g l a n d .
Biochim. Biophys. Acta, 141 (1967) 197-199
BBA 23344
Biosynthesis of quinolinic acid in a cell-free system It is well established that mammals, Neurospora and Xanthomonas pruni convert tryptophan to niacin 1. Escherichia coli and Bacillus subtilis2 and higher plants 3 do not possess the tryptophan-niacin pathway. The results of experiments with E. coli4, Mycobacterium tubercolosis n-7, Ricinus communis s and Nicotiana tobaccum 9 suggest that glycerol is a precursor of carbons 4, 5 and 6 of the pyridine ring of nicotinic acid and that a dicarboxylic acid is incorporated into carbons 2, 3 and 7 with carbon 7 being derived from a carboxyl group of the precursor acid. Recently, two reports which are not consistent with this pathway for the biosynthesis of nicotinic acid have appeared. SCOTT AND HUSSEY10 found that ill resting cells of Serratia marcescens [2-t4C~aspartate is converted to [7-14C~nicotinic acid and ISQUITH AND MOATn found that in a partially purified enzyme system from CIostridium butylicum aspartate, formate and acetate, but not glycerol, were incorporated into nicotinic acid. Biochim. Biophys. Acta, 141 (1967) 1 9 9 - 2 o i
200
SHORT COMMUNICATIONS
The recent discovery that quinolinic acid is converted to nicotinic acid mononucleotide in organisms which do not utilize the tryptophan-niacin pathway 12-14, strongly supports the hypothesis that quinolinate is an intermediate in niacin formation in organisms capable of using the glycerol-dicarboxylic acid pathway for pyridine ring biosynthesis. The results of the present investigation suggest that cellfree systems prepared from E. coli convert aspartate to carbons 2, 3, 7 and 8 of quinolinic acid. A mutant of E. coli lacking quinolinate phosphoribosyl transferase 15 (a gift of Dr. JOHN IMSANDE of Western Reserve University) was grown on a salts-glycerol medium 1~ containing IO-e M niacin. A cell-free extract was prepared by grinding 4 g of cells with 8 g of alumina, followed by suspension in 20 ml of 0.o2 M potassium phosphate buffer at pH 7.4 and centrifugation at 5000 × g for 30 rain. The reaction mixture for the biosynthesis of quinolinate contained 15 ml of the bacterial extract, uniformly labeled L-[14Claspartate, 5/~C (0.03/zmole); glycerol, 300 tzmoles; potassium phosphate pH, 7.4, IOOO/zmoles; MgC12, 20/zmoles; beef liver boiled juice, 6.5 ml; penicillin, 40000 units and streptomycin, 5 mg in a total volume of 25 ml. The reaction mixture was incubated at 37 ° for 5 h with shaking and deproteinized by heating to IOO°. After centrifugation, the supernatant solution was subjected to a Dowex I (formate) column chromatography as described by PREISS AND HANDLER17. Radioactivity of eluted fractions was monitered using a Tri-Carb liquid scintillation spectrometer. The radioactive peak eluting with 4 M formic acid was lyophilized and further purified by descending paper chromatography using: (I) propanol-water (80: 20); (2) e t h a n o l - I M ammonium acetate (7o:3o); and (3) butanol-acetic acidwater (4: I : 2). The compound which migrated with the same RF as authentic quinolinate in these solvent systems was further characterized by its reaction with purified quinolinate phosphoribosyltransferaselS; 4460 counts/rain of this compound were incubated for I h at 37 ° with 2. 4/zmoles phosphoribosylpyrophosphate, 2.0/,moles MgC12, 50/~moles potassium phosphate (pH 6.8) and 1.2 mg beef liver quinolinate phosphoribosyltransferase in a final volume of 4.0 ml. Approximately one-fourth the radioactivity in quinolinate was released as ~aco2 (997 counts/rain; assayed as previously described 15) by this enzymatic treatment. The other product of the reaction, nicotinic acid mononucleotide, was isolated by column chromatography ~7 and identified by its migration on paper chromatography in the solvent systems listed above (3818 counts/min). Another portion of the isolated quinolinic acid was pyrrolized to yield CO 2 and nicotinic acid. [14C~Quinolinate (2.35 m/zC) synthesized by the E. coti system in vitro and IO mg of carrier quinolinate were placed in a combustion tube of a Van Slyke wet combustion apparatus. The tube was heated at 19°0 for 30 rain in vacuo with an oil bath and the 14CO2 released from the a-carboxyl of quinolinic acid was collected directly into an ionization chamber and the radioactivity determined with a vibratingreed electrometer. Approximately one-fourth of the total activity of the quinolinic acid was recovered as laCO 2 (0.59 m/zC). The nicotinic acid formed was recovered from the walls of the combustion tube and a portion was decarboxylated as described by ORTEGA AND BROWN4. This method yields carbon 7 of nicotinic acid as CO 2 and the rest of the molecule as pyridine picrate. Two-thirds of the radioactivity (o.16o m/,C) from the decarboxylation of nicotinic acid was recovered as [l*C~pyridine picrate and one-third (0.084 m/,C) was recovered as CO v Biochim. Biophys. Acta, 141 (1967) 199--2Ol
SHORT COMMUNICATIONS
201
These results demonstrate that quinolinic acid can be synthesized in a cell-free system prepared from an E. coli mutant. Degradation of [14Clquinolinate formed from uniformly labeled [14C]aspartate showed that one-fourth of the radioactivity was located in the a-carboxyl group (C-7), one-fourth in the fl-carboxyl group (C-8) and one-half in the remainder of the molecule. This pattern is consistent with the direct incorporation of aspartate into carbons 2, 3, 7 and 8 of quinolinic acid, as shown in Scheme I.
H~i'--'C°°H -.---,--.~COOH 14~N-- "C 14 --'P COO 14
..~N/,,. eCOOFI
Scheme I
This research was supported in part by Grant No. GM-Io666 from the National Institute of Health and Grant Nos. GB-4645 and GB-5487 from the National Science Foundation.
Department of Biochemistry, Agricultural Experiment Station, Oklahoma State University, Stillwater, Okla. (U.S.A.) Department of Chemistry, California State College, Los Angeles, Calif. (U.S.A.)
N . 0GASAWARA
J. L. R. CHANDLER R. K. GHOLSON R. J. ROSSER A. J. ANDREOLI
I L. M. HENDERSON, R. K. GHOLSON AND C. E. DALGLIESH, in M. FLORKIN AND H. S. MASON, Comparative Biochemistry, Vol. 4, A c a d e m i c Press, N e w York, 1962, p. 241. 2 C. YANOFSKY, J. Bacteriol., 68 (1954) 577. 3 L . M . HENDERSON, J. F. SOMERASKI, D. R. RAO, P . H . L . W u , T. GRIFFITH AND R. U. BYERRUM, J. Biol. Chem., 234 (1959) 93. 4 M. V. ORTEGA AND G. M. BROWN, J. Biol. Chem., 235 (196o) 2939. 5 E. MOTHES, D. GROSS, H. R. SCH~ITTE AND K. MOTHES, Naturwissenschaften, 48 (1961) 623. 6 A. N. ALBERTSON AND A. G. MOAT, J. Bacteriol., 89 (1965) 54 o. 7 D. GROSS, H. R. SCHi)TTE, G. H~3BNER AND K. MOTHES, Tetrahedron Letters, No. 9 (1963) 541. 8 K. S. YANG AND G. lc{. WALLER, Phytoehemistry, 4 (1965) 881. 9 K. S. YANG, R. K. GHOLSON AND G. R. WALLER, J . Am. Chem. Soe., 87 (1965) 4185. io T. A. SCOTT AND H. HUSSEY, Bioehem. J., 96 (1965) 9C. II A. J. ISQUITHAND A. G. MOAT, Bioehem. Biophys. Res. Commun., 22 (1966) 565. 12 A. J. ANDREOLI, M. IKEDA, Y. •ISHIZUKA AND O. HAYAISHI, Bioehem. Biophys. Res. Commun., 12 (1963) 92. 13 L. A. HADWIGER, S. E. BADIEI, G. R. WALLER AND ]~-. K. GHOLSON, Biochem. Biophys. Res. Commun., 13 (1963) 466. 14 P. M. PACKMAN AND W. B. JAKOBY, Biochem. Biophys. Res. Commun., 18 (1965) 71o. 15 R. K. GHOLSON, I. UEDA, N. OGASAWARA AND L. M. HENDERSON, J. Biol. Chem., 239 (1964) 12o8. 16 R. A. YATES AND A. B. PARDEE, J. Biol. Chem., 221 (1956) 743. 17 J. PREISS AND P. HANDLER, J. Biol. Chem., 233 (1958) 488.
Received March I4th, 1967 Biochim. Biophys. ,4eta, 141 (1967) 199-2Ol
SHORT COMMUNICATIONS
boxylasen, TM when 14C0~. is introduced. However, malate remains labelled in C-4 in photosynthesising Atriplex leaves which suggests that little is formed by a second carboxylation with a labelled C 8 compound from the carbon-reduction cycle. Carbon fixed in the C-4 position of malate or aspartate cannot contribute directly to carbohydrate synthesis via known pathways. If this fixation is followed by transcarboxylation with another CO, acceptor, e.g. ribulose 1,5-diphosphate, as suggested in sugar canO, labile malate and aspartate pools in the chloroplast is may serve as a temporary store of carbon. The high affinity for CO, of such a carboxylation system may reasonably explain photosynthetic carbon fixation at physiological CO s concentrations.
Botany Department, University of Adelaide, Adelaide (Australia)
C. B.
OSMOND*
I M. CALVIN AND J. A. BASSHAM, The Photosynthesis of Carbon Compounds, B e n j a m i n , N e w York, 1962. 2 H. P. KORTSCHAK, C. E. HARTT AND G. O. BURR, Plant Physiol., 4 ° (1965) 209. 3 M. D. HATCH AND C. R. SLACK, Biochem. J., IOi (1966) lO 3. 4 M. D. HATCH, C. R. SLACK AND H. S. JOHNSON, Biochem. J., lO2 (1967) 417 . 5 S. ARONOFF, Techniques of Radiobioehemistry, I o w a S t a t e U n i v e r s i t y Press, A m e s , 1956. 6 P. M. NOSSAL, Biochem. J., 49 (1951) 4o7. 7 L. R. ROSENBERG, J. B. CAPINDALE AND F. R. WHATLEY, Nature, 18I (1958) 632. 8 C. B. OSMOND AND P. N. AVADAHNI, in p r e p a r a t i o n . 9 A. A. BENSON, S. KAWANCHI, P. HAYES AND M. CALVIN, J. Am. Chem. Sot., 74 (1952) 4477. io D. GRAHAM AND C. P. WITTINGHAM, in p r e p a r a t i o n . II D. A. WALKER, Biol. Rev., 37 (1962) 215. 12 E. RACKER, Arch. Biochem. Biophys., 69 (1957) 3oo. 13 A. OGUN AND C. R. STOCKING, Plant Physiol., 4 ° (1965) 825.
Received March 6th, 1967 * P r e s e n t a d d r e s s : B o t a n y D e p a r t m e n t , U n i v e r s i t y of C a m b r i d g e , Cambridge, E n g l a n d .
Biochim. Biophys. Acta, 141 (1967) 197-199
BBA 23344
Biosynthesis of quinolinic acid in a cell-free system It is well established that mammals, Neurospora and Xanthomonas pruni convert tryptophan to niacin 1. Escherichia coli and Bacillus subtilis2 and higher plants 3 do not possess the tryptophan-niacin pathway. The results of experiments with E. coli4, Mycobacterium tubercolosis n-7, Ricinus communis s and Nicotiana tobaccum 9 suggest that glycerol is a precursor of carbons 4, 5 and 6 of the pyridine ring of nicotinic acid and that a dicarboxylic acid is incorporated into carbons 2, 3 and 7 with carbon 7 being derived from a carboxyl group of the precursor acid. Recently, two reports which are not consistent with this pathway for the biosynthesis of nicotinic acid have appeared. SCOTT AND HUSSEY10 found that ill resting cells of Serratia marcescens [2-t4C~aspartate is converted to [7-14C~nicotinic acid and ISQUITH AND MOATn found that in a partially purified enzyme system from CIostridium butylicum aspartate, formate and acetate, but not glycerol, were incorporated into nicotinic acid. Biochim. Biophys. Acta, 141 (1967) 1 9 9 - 2 o i
200
SHORT COMMUNICATIONS
The recent discovery that quinolinic acid is converted to nicotinic acid mononucleotide in organisms which do not utilize the tryptophan-niacin pathway 12-14, strongly supports the hypothesis that quinolinate is an intermediate in niacin formation in organisms capable of using the glycerol-dicarboxylic acid pathway for pyridine ring biosynthesis. The results of the present investigation suggest that cellfree systems prepared from E. coli convert aspartate to carbons 2, 3, 7 and 8 of quinolinic acid. A mutant of E. coli lacking quinolinate phosphoribosyl transferase 15 (a gift of Dr. JOHN IMSANDE of Western Reserve University) was grown on a salts-glycerol medium 1~ containing IO-e M niacin. A cell-free extract was prepared by grinding 4 g of cells with 8 g of alumina, followed by suspension in 20 ml of 0.o2 M potassium phosphate buffer at pH 7.4 and centrifugation at 5000 × g for 30 rain. The reaction mixture for the biosynthesis of quinolinate contained 15 ml of the bacterial extract, uniformly labeled L-[14Claspartate, 5/~C (0.03/zmole); glycerol, 300 tzmoles; potassium phosphate pH, 7.4, IOOO/zmoles; MgC12, 20/zmoles; beef liver boiled juice, 6.5 ml; penicillin, 40000 units and streptomycin, 5 mg in a total volume of 25 ml. The reaction mixture was incubated at 37 ° for 5 h with shaking and deproteinized by heating to IOO°. After centrifugation, the supernatant solution was subjected to a Dowex I (formate) column chromatography as described by PREISS AND HANDLER17. Radioactivity of eluted fractions was monitered using a Tri-Carb liquid scintillation spectrometer. The radioactive peak eluting with 4 M formic acid was lyophilized and further purified by descending paper chromatography using: (I) propanol-water (80: 20); (2) e t h a n o l - I M ammonium acetate (7o:3o); and (3) butanol-acetic acidwater (4: I : 2). The compound which migrated with the same RF as authentic quinolinate in these solvent systems was further characterized by its reaction with purified quinolinate phosphoribosyltransferaselS; 4460 counts/rain of this compound were incubated for I h at 37 ° with 2. 4/zmoles phosphoribosylpyrophosphate, 2.0/,moles MgC12, 50/~moles potassium phosphate (pH 6.8) and 1.2 mg beef liver quinolinate phosphoribosyltransferase in a final volume of 4.0 ml. Approximately one-fourth the radioactivity in quinolinate was released as ~aco2 (997 counts/rain; assayed as previously described 15) by this enzymatic treatment. The other product of the reaction, nicotinic acid mononucleotide, was isolated by column chromatography ~7 and identified by its migration on paper chromatography in the solvent systems listed above (3818 counts/min). Another portion of the isolated quinolinic acid was pyrrolized to yield CO 2 and nicotinic acid. [14C~Quinolinate (2.35 m/zC) synthesized by the E. coti system in vitro and IO mg of carrier quinolinate were placed in a combustion tube of a Van Slyke wet combustion apparatus. The tube was heated at 19°0 for 30 rain in vacuo with an oil bath and the 14CO2 released from the a-carboxyl of quinolinic acid was collected directly into an ionization chamber and the radioactivity determined with a vibratingreed electrometer. Approximately one-fourth of the total activity of the quinolinic acid was recovered as laCO 2 (0.59 m/zC). The nicotinic acid formed was recovered from the walls of the combustion tube and a portion was decarboxylated as described by ORTEGA AND BROWN4. This method yields carbon 7 of nicotinic acid as CO 2 and the rest of the molecule as pyridine picrate. Two-thirds of the radioactivity (o.16o m/,C) from the decarboxylation of nicotinic acid was recovered as [l*C~pyridine picrate and one-third (0.084 m/,C) was recovered as CO v Biochim. Biophys. Acta, 141 (1967) 199--2Ol
SHORT COMMUNICATIONS
201
These results demonstrate that quinolinic acid can be synthesized in a cell-free system prepared from an E. coli mutant. Degradation of [14Clquinolinate formed from uniformly labeled [14C]aspartate showed that one-fourth of the radioactivity was located in the a-carboxyl group (C-7), one-fourth in the fl-carboxyl group (C-8) and one-half in the remainder of the molecule. This pattern is consistent with the direct incorporation of aspartate into carbons 2, 3, 7 and 8 of quinolinic acid, as shown in Scheme I.
H~i'--'C°°H -.---,--.~COOH 14~N-- "C 14 --'P COO 14
..~N/,,. eCOOFI
Scheme I
This research was supported in part by Grant No. GM-Io666 from the National Institute of Health and Grant Nos. GB-4645 and GB-5487 from the National Science Foundation.
Department of Biochemistry, Agricultural Experiment Station, Oklahoma State University, Stillwater, Okla. (U.S.A.) Department of Chemistry, California State College, Los Angeles, Calif. (U.S.A.)
N . 0GASAWARA
J. L. R. CHANDLER R. K. GHOLSON R. J. ROSSER A. J. ANDREOLI
I L. M. HENDERSON, R. K. GHOLSON AND C. E. DALGLIESH, in M. FLORKIN AND H. S. MASON, Comparative Biochemistry, Vol. 4, A c a d e m i c Press, N e w York, 1962, p. 241. 2 C. YANOFSKY, J. Bacteriol., 68 (1954) 577. 3 L . M . HENDERSON, J. F. SOMERASKI, D. R. RAO, P . H . L . W u , T. GRIFFITH AND R. U. BYERRUM, J. Biol. Chem., 234 (1959) 93. 4 M. V. ORTEGA AND G. M. BROWN, J. Biol. Chem., 235 (196o) 2939. 5 E. MOTHES, D. GROSS, H. R. SCH~ITTE AND K. MOTHES, Naturwissenschaften, 48 (1961) 623. 6 A. N. ALBERTSON AND A. G. MOAT, J. Bacteriol., 89 (1965) 54 o. 7 D. GROSS, H. R. SCHi)TTE, G. H~3BNER AND K. MOTHES, Tetrahedron Letters, No. 9 (1963) 541. 8 K. S. YANG AND G. lc{. WALLER, Phytoehemistry, 4 (1965) 881. 9 K. S. YANG, R. K. GHOLSON AND G. R. WALLER, J . Am. Chem. Soe., 87 (1965) 4185. io T. A. SCOTT AND H. HUSSEY, Bioehem. J., 96 (1965) 9C. II A. J. ISQUITHAND A. G. MOAT, Bioehem. Biophys. Res. Commun., 22 (1966) 565. 12 A. J. ANDREOLI, M. IKEDA, Y. •ISHIZUKA AND O. HAYAISHI, Bioehem. Biophys. Res. Commun., 12 (1963) 92. 13 L. A. HADWIGER, S. E. BADIEI, G. R. WALLER AND ]~-. K. GHOLSON, Biochem. Biophys. Res. Commun., 13 (1963) 466. 14 P. M. PACKMAN AND W. B. JAKOBY, Biochem. Biophys. Res. Commun., 18 (1965) 71o. 15 R. K. GHOLSON, I. UEDA, N. OGASAWARA AND L. M. HENDERSON, J. Biol. Chem., 239 (1964) 12o8. 16 R. A. YATES AND A. B. PARDEE, J. Biol. Chem., 221 (1956) 743. 17 J. PREISS AND P. HANDLER, J. Biol. Chem., 233 (1958) 488.
Received March I4th, 1967 Biochim. Biophys. ,4eta, 141 (1967) 199-2Ol