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The enzymic and chemical formation of 3,4-dihydro-3,4-dihydroxybenzoic acid: A new compound derived from chorismic acid
I82
PRELIIEINARY NOTES
~BA 21243
The enzymic and chemical formation of 3,4-dihyd~'o-3,4-dihydro×yben~oic acid: ~ new compound d e r ~ e d from chef,stoic add Chorismic acid (I) is a branch point compound in the biosynthesis o~ the are matic amino acids and vitami~sL ic has now been shox~ that 3,4-dihydro-3,4--dihydroxybenzoic acid can be formed from chorismic acid (Fig. z) either by acid treatm e n t or enzymically using cell extracts from A efobacte~, aeroge~es. COOH 3
coo~
c~
/
OOH I
~o. i[~
~OOH
. OH o~ ~
H
~
COOH
OH }V Fig. ~. T h e c o n v e r s i o n of chorismic acid (!] into 3 , 4 - d i h y d r o - 3 , 4 - d i h y d r o x y b e n z o i c acid (II) b v h e a t i n g w i t h dilute acid, or enzymically, a n d t h e c h e m i c a l d e c o m p o s i t i o n of (!I] into 3 - h y d r o x y benzoic acid (I1[) a n d 4 - h y d r o x y b e n z o i c acid (IV).
Chorismic acid (o.9 g) was heated at Ioo ° for 15 rain in 0.o5 M HC1 giving a mixture of phenylpyruvic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid and a new compound. Chromatography on Dowex z (CI-/ and elution with I M NH ~C! at pH 8, foilowed by continuous ether extraction of the appropriate fractions, and subsequent crystallization yielded about 8o mg of a white crvstalline compound (m.p. I7 o°, decomp.). The new compound was identified as 3,4-dihydro-3,4-dihydroxybenzoic acid (H~ from NY~R and mass spectra. The NMR spectrum of the new compound in deuterodimethylsulphoxide is similar to that of chorismic acid in the same solvent except that the two doublets from the methylene group of the pyruvyl moiety of chorismic acid are absent. The similarity of the spectra indicates that the ring sys'cem of the new compound is similar to that of chorismic acid. The ultraviolet absorption spectra are also similar: chorismic acid, 2max 275 nm (e 263o); the new compound. 2raax 272 nm (e 225o). The mass spectrum of the new compound is very slmiiar co that of 2,3-dihydro-2,3-dihydroxybenzoic acid 2, and the accurate mass of the moiecu!ar ion at m/e I56 was found to be I56.o43o (C~HsO~ reeuires I56.o432 ). The new compotmd is readily distinguished from 2,3~dihydro-2,3-dihydroxybenzoic acid by highvoltage paper electrophoresis. Heating the new compound in ~ M HCI at ~roo° gave 3-hydroxybenzoic acid and a small amonnt of 4-hydroxybenzoic acid (Fig. I~. The absolute stereochemistry of 3,4-dihydro-3,4-dihydroxybenzoic acid (Fig. z was determined by ozo~olysis followed by oxidative decomposition of the ozonide to yield tartaric acid. The tartaric acid so obtained was identified as (--~tar~aric acid (S,S) by optical rotatory dispersion spectroscopy. The 3,4-substituents are therefore tfa~s to each other and in the R,R absolute configuration. Thns the absotute configuration of chorismic acid ~ has been retained. Biochim. Biophys. Acts, 177 (I969) I 8 2 - x 8 3
PREL][M[NAIRY N O T E S
18 3
3,4-Dihydro-3,4-dihydroxybenzoic acid was also found to be formed enzymically from chorismic acid. Cell extracts were prepared from A. aerogenes grown in an irondeficient medium. The incubation of chorismate and cell extract in buffer yielded a mixture of 2,3-dihydro-2,3-dihydroxybenzoic~ and 3,4-dihydro-3,4-dihydroxybenzoic acids. The latter compound was purified by chromatography on Dowex I followed b y high-voltage paper electrophoresis and crystallization. The ultraviolet, mass, infrared and optical rotatory dispersion spectra of the enzymically produced material were identical with the spectra of the 3,4-dihydro-3,4-dihydroxybenzoic acid isolated after heating chorismic acid. The enzyme(s) forming 3,4-dihydro-3,4-dihydroxybenzoic acid is repressed b y iron:, as are the enzymes involved in the biosynthesis of 2,3-dihydroxybenzoate~, indicating that the new compound may play some part, directly or indirectly, in metal metabolism. 3,4-Dihydro-3,4-dihydroxybenzoic acid has a slight sparing effect on the 2,3-dihydroxybenzoate requirement of a multiple aromatic auxotroph of Escherichia coli~ although it does not promote growth alone. This is consistent with the observation that it is not converted to 2,3-dihydroxybenzoic acid by cell extracts of A. aerogenes or E. coli under conditions used to convert 2,3-dihydro-2,3-dihydroxybenzoic acid into 2,3-dihydroxybenzoate~. The new compound was not active as a precursor of 4-hydroxybenzoate or 4-aminobenzoate when tested with cell extracts of A. aerogenes capable of converting chorismate to these compounds. In addition, no evidence has yet been obtained that the new compound serves as a precursor of 3,4-dihydroxybenzoic acid which, in A. aerogenes (unpuNished observations), as in other organisms~, ~, is formed from dehydroshikimic acid. We wish to thank Mr. J. Lee and Mrs. J. McDonald for technical assistance, Mr. C. G. Macdonald for mass spectrometry, and Dr. T. Batterham for assistance in interpreting the NMR spectra.
Biochemistry Departme~#, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. (Australia) I 2 3 4 5 6 7
I. G. Y o u ~ c F. GIBSON
M. I. GIBSON AND F. GIBSON, Biochem. J., 9o (1964) 248. I. G. YOUNG, L. M. JACKMANAND F. GIBSON, in p r e p a r a t i o n . J- M. EDWARDS AND L. M. JACKMAN, Australian J. Chem., 18 (1965) 1227. I. G. YOUNG, L. ~/~. JACKMAN AND F. GIBSON, Biochim. Biophys. Acta, 148 (1967) 313 . I. G. YOUNG, G. ]3. C o x AnD F. G i b s o n , Bioehim. Biophys. Acta, 141 (I967) 319. S. 1R. GRoss, j~. Biol. Chem., 233 (1958) 1146. J. L. C£~'OVAS, M. L. WH~ELIS AND R. Y. STANIER, European J. Biochem., 3 (1968) 293.
Received October 28th, 1968 Biochim. Biophys. Acta, 177 (1969) 182-183
PRELIIEINARY NOTES
~BA 21243
The enzymic and chemical formation of 3,4-dihyd~'o-3,4-dihydro×yben~oic acid: ~ new compound d e r ~ e d from chef,stoic add Chorismic acid (I) is a branch point compound in the biosynthesis o~ the are matic amino acids and vitami~sL ic has now been shox~ that 3,4-dihydro-3,4--dihydroxybenzoic acid can be formed from chorismic acid (Fig. z) either by acid treatm e n t or enzymically using cell extracts from A efobacte~, aeroge~es. COOH 3
coo~
c~
/
OOH I
~o. i[~
~OOH
. OH o~ ~
H
~
COOH
OH }V Fig. ~. T h e c o n v e r s i o n of chorismic acid (!] into 3 , 4 - d i h y d r o - 3 , 4 - d i h y d r o x y b e n z o i c acid (II) b v h e a t i n g w i t h dilute acid, or enzymically, a n d t h e c h e m i c a l d e c o m p o s i t i o n of (!I] into 3 - h y d r o x y benzoic acid (I1[) a n d 4 - h y d r o x y b e n z o i c acid (IV).
Chorismic acid (o.9 g) was heated at Ioo ° for 15 rain in 0.o5 M HC1 giving a mixture of phenylpyruvic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid and a new compound. Chromatography on Dowex z (CI-/ and elution with I M NH ~C! at pH 8, foilowed by continuous ether extraction of the appropriate fractions, and subsequent crystallization yielded about 8o mg of a white crvstalline compound (m.p. I7 o°, decomp.). The new compound was identified as 3,4-dihydro-3,4-dihydroxybenzoic acid (H~ from NY~R and mass spectra. The NMR spectrum of the new compound in deuterodimethylsulphoxide is similar to that of chorismic acid in the same solvent except that the two doublets from the methylene group of the pyruvyl moiety of chorismic acid are absent. The similarity of the spectra indicates that the ring sys'cem of the new compound is similar to that of chorismic acid. The ultraviolet absorption spectra are also similar: chorismic acid, 2max 275 nm (e 263o); the new compound. 2raax 272 nm (e 225o). The mass spectrum of the new compound is very slmiiar co that of 2,3-dihydro-2,3-dihydroxybenzoic acid 2, and the accurate mass of the moiecu!ar ion at m/e I56 was found to be I56.o43o (C~HsO~ reeuires I56.o432 ). The new compotmd is readily distinguished from 2,3~dihydro-2,3-dihydroxybenzoic acid by highvoltage paper electrophoresis. Heating the new compound in ~ M HCI at ~roo° gave 3-hydroxybenzoic acid and a small amonnt of 4-hydroxybenzoic acid (Fig. I~. The absolute stereochemistry of 3,4-dihydro-3,4-dihydroxybenzoic acid (Fig. z was determined by ozo~olysis followed by oxidative decomposition of the ozonide to yield tartaric acid. The tartaric acid so obtained was identified as (--~tar~aric acid (S,S) by optical rotatory dispersion spectroscopy. The 3,4-substituents are therefore tfa~s to each other and in the R,R absolute configuration. Thns the absotute configuration of chorismic acid ~ has been retained. Biochim. Biophys. Acts, 177 (I969) I 8 2 - x 8 3
PREL][M[NAIRY N O T E S
18 3
3,4-Dihydro-3,4-dihydroxybenzoic acid was also found to be formed enzymically from chorismic acid. Cell extracts were prepared from A. aerogenes grown in an irondeficient medium. The incubation of chorismate and cell extract in buffer yielded a mixture of 2,3-dihydro-2,3-dihydroxybenzoic~ and 3,4-dihydro-3,4-dihydroxybenzoic acids. The latter compound was purified by chromatography on Dowex I followed b y high-voltage paper electrophoresis and crystallization. The ultraviolet, mass, infrared and optical rotatory dispersion spectra of the enzymically produced material were identical with the spectra of the 3,4-dihydro-3,4-dihydroxybenzoic acid isolated after heating chorismic acid. The enzyme(s) forming 3,4-dihydro-3,4-dihydroxybenzoic acid is repressed b y iron:, as are the enzymes involved in the biosynthesis of 2,3-dihydroxybenzoate~, indicating that the new compound may play some part, directly or indirectly, in metal metabolism. 3,4-Dihydro-3,4-dihydroxybenzoic acid has a slight sparing effect on the 2,3-dihydroxybenzoate requirement of a multiple aromatic auxotroph of Escherichia coli~ although it does not promote growth alone. This is consistent with the observation that it is not converted to 2,3-dihydroxybenzoic acid by cell extracts of A. aerogenes or E. coli under conditions used to convert 2,3-dihydro-2,3-dihydroxybenzoic acid into 2,3-dihydroxybenzoate~. The new compound was not active as a precursor of 4-hydroxybenzoate or 4-aminobenzoate when tested with cell extracts of A. aerogenes capable of converting chorismate to these compounds. In addition, no evidence has yet been obtained that the new compound serves as a precursor of 3,4-dihydroxybenzoic acid which, in A. aerogenes (unpuNished observations), as in other organisms~, ~, is formed from dehydroshikimic acid. We wish to thank Mr. J. Lee and Mrs. J. McDonald for technical assistance, Mr. C. G. Macdonald for mass spectrometry, and Dr. T. Batterham for assistance in interpreting the NMR spectra.
Biochemistry Departme~#, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. (Australia) I 2 3 4 5 6 7
I. G. Y o u ~ c F. GIBSON
M. I. GIBSON AND F. GIBSON, Biochem. J., 9o (1964) 248. I. G. YOUNG, L. M. JACKMANAND F. GIBSON, in p r e p a r a t i o n . J- M. EDWARDS AND L. M. JACKMAN, Australian J. Chem., 18 (1965) 1227. I. G. YOUNG, L. ~/~. JACKMAN AND F. GIBSON, Biochim. Biophys. Acta, 148 (1967) 313 . I. G. YOUNG, G. ]3. C o x AnD F. G i b s o n , Bioehim. Biophys. Acta, 141 (I967) 319. S. 1R. GRoss, j~. Biol. Chem., 233 (1958) 1146. J. L. C£~'OVAS, M. L. WH~ELIS AND R. Y. STANIER, European J. Biochem., 3 (1968) 293.
Received October 28th, 1968 Biochim. Biophys. Acta, 177 (1969) 182-183