- Email: [email protected]
[18] Synthesis of 17O- or 18O-Enriched dihydroxy aromatic compounds
[18]
SYNTHESIS OF DIHYDROXY AROMATIC COMPOUNDS
107
both the C-3 and C-4 positions are also turned over, but at dramatically reduced rates (< 10% that ofgentisate). All three main functional groups of gentisate appear to be required for efficient turnover, in that little or no turnover is observed with compounds in which any one of these groups is substituted with another functionality. Additionally, monosubstituted benzoates such as salicylate, 3-hydroxybenzoate, and thiosalicylate are not turned over but are relatively good inhibitors of the enzyme. Gentisate 1,2-dioxygenase is inactivated in a time- and concentration-dependent manner by oxidants such as H202 and K3Fe(CN)6. Partial reactivation is effected by ascorbate. Acknowledgment This work was supported by a grant from the National Institutes of Health (GM 24689).
[18] S y n t h e s i s o f 170- o r 1 s O - E n r i c h e d D i h y d r o x y Aromatic Compounds B y A L L E N M . ORVILLE, M A R K R . H A R P E L , a n d JOHN D . LIPSCOMB
Specific isotopic labeling of the oxygen atoms of phenolic and catecholic compounds is of use in spectroscopic and kinetic investigations of a variety of enzymes that bind these molecules as substrates or inhibitors.t.2 A convenient and versatile synthetic procedure is depicted in Scheme 1. The position of incorporation of the isotopically enriched oxygen atom is determined by the source of the enriched oxygen (H20 or 02) and the position of substituents in the starting aromatic amine. This methodology is illustrated here with the synthesis of isotopically enriched homoprotocatechuate (3,4-dihydroxyphenylacetate, HPCA a) as an example. This procedure has appeared in a previous report. 2 These techniques have been shown to be applicable to a wide variety of phenolic and catecholic aromatic compounds (Table I).
1D. M. Arciero and J. D. Lipscomb, J. Biol. Chem. 261, 2170 (1986). 2 A. M. Orville and J. D. Lipscomb, J. Biol. Chem. 264, 8791 (1989). 3 Abbreviations used are given in Table I.
METHODS IN ENZYMOLOGY,VOL. 188
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108
HYDROCARBONS AND RELATED COMPOUNDS
[ 18]
O"
R
I
R Diazonium Reaction
N2
R
02
OH 2
I
Hydroxylation
R
SCHEME 1
Chemical Methods
Diazonium Reaction: Synthesis 0f[170]- or [1sO]Hydroxyl-Enriched 4-Hydroxyphenylacetate Reagents 4-Aminophenylacetate (4APA), recrystallized from hot water Distilled 170- or toO-enriched water Desiccated NaNO2 Procedure. In this procedure an aromatic amine is converted to the corresponding diazonium salt and then hydrolyzed in ~0- or toO-enriched water to form the labeled phenol. The method was adapted from that of Robertson and Jacobs. 4 Formation of the diazonium salt. Add 50 mg of 4APA (1.0 molar equivalent) to a small vial containing 600/tl 170- or toO-enriched water. Cool the mixture in an ice bath with stirring and add 2.2 molar equivalents of 12 N HC1 (~ 60/tl). In a separate vial, add approximately 100 ~1170- or toO-enriched water to 1.1 molar equivalents of NaNO2 (25 mg) and cool on ice. Add the NaNO2 solution to the amine in 5-/A aliquots from a cold Hamilton syringe over 30 rain. Five minutes after the last addition of the NaNO2 add about 50 mg urea to remove the excess HONO. Hydrolysis of the diazonium salt. Heat, with continuous stirring 170- or mSO-endched water (600/A) containing 2.2 molar equivalents 12 N HC1 (-60/~1) to 90-100 ° in a two-neck pear-shaped reaction flask (10 ml) immersed in an oil bath. The vertical neck of the flask (T 14/20) is connected to a condenser, and the side neck (T10/18) is sealed with a rubber septum. Add 25-/tl aliquots of the cold diazonium salt solution through the septum with a cold syringe. Allow the evolution of N2 to subside between additions. Continue heating for 15 min after the final addition. Partial 4 G. R. Robertson and T. L. Jaeobs, in "Laboratory Practice of Organic Chemistry," 4th Ed. Macmillan, New York, 1962.
0
2, I-
N
O a~ r~
0
O
~~ ~
110
HYDROCARBONS AND RELATED COMPOUNDS
[ 18]
exchange of the carboxyl oxygens of some aromatic acids with water may occur during this step of the procedure. This exchange can be reversed as discussed in the carboxyl exchange section below. Recovery oflZO- or ~SO-enriched water. Cool the reaction to 0 °, drain the condenser, and connect the reaction flask to a short-arm vacuum distillation curve. Connect the other end of the curve to a 10-ml pearshaped collection flask. Cool both the reaction flask and the collection flask in liquid nitrogen, draw a vacuum, and then isolate the system from the vacuum line. On warming the reaction flask to 23 °, -the ~70- or nO_ enriched water is removed by distillation and is condensed in the cold collection flask. The enriched water recovered in this way must be neutralized, treated with activated charcoal, and distilled again before reuse.
Isolation of [~zO] or [1sO]hydroxyl-enriched 4-hydroxyphenylacetate. Dissolve the lyophilized hydrolysis product in 1 ml of water containing 1.2 molar equivalents of 6 N NaOH. The mixture is then treated with activated charcoal and filtered into a vial containing 5 equivalents of HC1. Saturate the filtrate with NaC1 and extract it several times with l ml of anhydrous diethyl ether. Combine the ether extracts and dry them with anhydrous MgSO 4. After filtering, evaporate the solvent under a stream of argon or by vacuum distillation. The product is pure, with an isotopic enrichment essentially the same as that of the enriched water used in the hydrolysis. This reaction proceeds readily with aniline and m- or p-amino-substituted benzoates or phenylacetates. The diazonium reaction does not produce the desired phenolic products in appreciable yields from aromatic compounds with substituents ortho to the amine.
Carboxyl Oxygen Exchange: Synthesis of [170] or [IsO]Carboxyl-Enriched Homoprotocatechuate Reagents Recrystallized HPCA Distilled ~70- or ~80-enriched water Argon or nitrogen gas passed over a column of BASF copper catalyst (Kontes, Vineland, NJ, 655960) to remove traces of contaminating 02 Procedure. The carboxylate oxygens of aromatic acids are exchanged with 170- or ~SO-enriched water via an acid-catalyzed reaction. To HPCA (50 mg) in a thick-walled hydrolysis tube (bulb v o l u m e - 1 ml) add enriched or unenriched water (550/d) and 12 N HC1 (50/11). Draw a vacuum and refill the tube with oxygen-free argon or nitrogen several times to achieve anaerobiosis. Freeze the sample, then evacuate and seal the
[18]
SYNTHESIS OF DIHYDROXY AROMATIC COMPOUNDS
II1
tube. The sealed tube is placed in an oven at 110 ° for 24 hr during which time the exchange occurs. After cooling, the sample is frozen and the tube opened. The 170- or 180-enriched water is recovered by distillation as discussed above. Mass spectral analysis of the [170] or [180]carboxylenriched HPCA shows complete exchange. Benzoate, phenylacetate, and their m- and/or p-hydroxyl-substituted derivatives yield carboxyl oxygenenriched compounds with this procedure. In the case of o-hydroxyl-substituted benzoates and phenylacetates, the incorporation is not limited exclusively to the carboxyl oxygens under the conditions described here. Conditions that promote the carboxyl oxygen exchange are briefly achieved during the hydrolysis of the diazonium salt discussed above. Undesired carboxyl oxygen enrichment can be eliminated without affecting the enrichment of the hydroxyl oxygen atom by carrying out the exchange reaction in unenriched water. Enzymatic Methods
Assay Methods Catecholic dioxygenase and aromatic hydroxylase activity assays have been described in this and earlier volumes of this series.5-s
Microbial Induction Monooxygenases of the type required for the right half of Scheme 1 are expressed by many bacteria when these organisms are cultured with the monohydroxy aromatic compound as the sole source of carbon and energy (Table I). 1,2,8-11 Several of these have been purified to homogeneity, and at least one is commercially availableJ 2 Growth of the Organism. Brevibacterium fuscum (ATCC 15933) is cultured in the medium described elsewhere in this volume5 with alteration in the carbon source as indicated below. Transfer a loop filled with cells from an agar plate containing medium plus 3 g/liter 4HBA to a 300-ml flask containing 100 ml of the same medium and carbon source. After 5 j. W. Whittaker, A. M. Orville, and J. D. Lipscomb, this volume [14]. 6 H. Fujisawa, this series, Vol. 17A, p. 526. 7 H. Kita and S. Senoh, this seres, Vol. 17A, p. 645. s M. Husain, L. M. Schopfer, and V. Massey, this series, Vol. 53, p. 543. 9 j. L. Michalover and D. W. Ribbons, Biochem. Biophys. Res. Commun. 55, 888 (1973). l°E. R. Blakley, W. Kurz, H. Halvorson, and F. J. Simpson, Can. J. Microbiol. 13, 147 (1967). 11 L-H. Wang, R. Y. Hamzah, Y. Yu, and S-C. Tu, Biochemistry 26, 1099 (1987). 124-Hydroxybenzoate 3-hydroxylase is available from Sigma Chemical Co., St. Louis, MO.
112
HYDROCARBONS AND RELATED COMPOUNDS
[18]
14 hr of growth on a rotary shaker at 30", transfer 5 ml of the culture to a 2-liter flask containing 1 liter of medium with a carbon source of 2.0 g 4HBA and 1.0 g 4HPA. After 14 hr of growth, transfer the culture to an 18-liter carboy containing 15 liters of medium with 2 g/liter 4HPA as the only carbon source. The culture is vigorously aerated ( - 1 liter/see), until the end of log phase growth ( - 24- 30 hr) with the addition of 2.0 g/liter of neutralized 4HPA after 12 hr. The cells are harvested with a water-cooledcontinuous-flow centrifuge, frozen rapidly in powdered dry ice, and stored at -80*. The yield is approximately 5 g of cells (wet weight) per liter of medium. Under these conditions, the 4HPA 3-hydroxylase and the catecholic dioxygenases protocatechuate 3,4-dioxygenase (3,4-PCD) and homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) are induced.
Purification of 4-Hydroxyphenylacetate 3-Hydroxylase Reagents MOPS buffer: 50 m M 4-morpholinopropanesulfonic acid, adjusted to pH 7.0 with 6 N NaOH Phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, MO), toxic Ribonuclease and deoxyribonuclease (Sigma) Enzyme-grade ammonium sulfate (Schwarz Mann Chemical Co., Cleveland, OH) Flavin adenine dinucleotide (FAD) (Sigma) A partial purification of 4HPA 3-hydroxylase is described here. The primary objective of this preparation procedure is to separate the hydroxylase from the catecholic dioxygenases that would degrade the reaction product during the extended in vitro enzymatic hydroxylation reaction.
Step 1: Preparation of Cell-Free Extract. Frozen cells (50 g) are hammered into a fine meal and rapidly suspended in 100 ml of MOPS buffer containing 2 m M PMSF, l0 m M EDTA, 1 m M 2-mercaptoethanol (2-ME), 10/~M FAD, and 2 mg each ribonuclease and deoxyribonuclease. Sonicate the cells for 45 min at 80% power with a Branson Model 350 sonicator equipped with a 3/4-inch fiat tip. The temperature is maintained at 5-10*with a 2-propanol-dry ice bath. Remove the cell debris by centrifugation for 30 min at 22,000 g and 4". Step 2: Ammonium Sulfate Fractionation. The cell-free extract is made 55% saturated in ammonium sulfate, the pH is adjusted to 7.0 with 6 N NaOH, and the solution is centrifuged as above. All of the 3,4-PCD and approximately half of the 2,3-HPCD is found in the pellet. Bring the supernatant to 70% saturation in ammonium sulfate (pH 7.0) to precipitate the 4HPA 3-hydroxylase and residual 2,3-HPCD and centrifuge.
[ 18]
SYNTHESIS OF DIHYDROXY AROMATICCOMPOUNDS
113
Step 3: Ion-Exchange Chromatography. Dissolve the pellet in 10 ml of MOPS buffer containing 2 m M PMSF and centrifuge at 22,000 g and 4 ° for 15 min to remove any insoluble protein. Dilute the supernatant to 200 ml and load it onto a column of Whatman DE-52 (3 × 18 cm) equilibrated with buffer. After washing with 75 m M ammonium sulfate in buffer, elute the residual 2,3-HPCD with a linear gradient (100 and 100 ml) from 75 to 150 m M ammonium sulfate in buffer. Elute the 4HPA 3-hydroxylase activity with a linear gradient (250 and 250 ml) from 150 to 300 m M ammonium sulfate in buffer. Fractions with more than 0.5 units/ ml hydroxylase activity are pooled and reactivated (-400%) by incubation with 1 m M 2-ME and 10/tM FAD for 20 min. Step 4: Concentration and Storage. Precipitate the reactivated hydroxylase by bringing the solution to 70% saturation in ammonium sulfate (pH 7.0). After centrifugation, redissolve the pellet in 3 ml of MOPS buffer containing 1 m M 2-ME and 10/zM FAD. Typically this preparation has approximately 70 units/ml and a specific activity of about 20 units/mg protein. The enzyme is frozen with liquid N2 and can be stored at 77 K for several months without loss of activity. Synthesis of [~70]- or [1sO]Hydroxyl-Enriched Homoprotocatechuate Reagents Buffer, 0.25 M MOPS, pH 7.0, containing 1 m M 2-ME and 10 FAD Glucose 6-phosphate (G6P), 50 m M 4HPA, 25 mM, unenriched or [170]- or [180]hydroxyl-enriched NADPH, 0.6 m M 170- or 1SO-enriched or unenriched 02 Glucose-6-phosphate dehydrogenase (G6PDH) (Sigma) 4HPA 3-hydroxylase Monitoring Hydroxylase Reaction Progress. The extended in vitro hydroxylase reaction progress can be followed in most cases by monitoring the loss of the monohydroxyaromatic substrate and/or increase in dihydroxyaromatic product with a UV spectrophotometer. However, the electronic spectra of 4HPA (2m~ 278 nm, ~ 1400 M -1 cm -1) and HPCA ( 2 ~ 282 nm, e 2500 M -~ cm -t) do not permit simple independent quantitations in a complex mixture. Determination of the product concentration using HPLC is a suitable alternative. HPLC Determinations. The column is a Beckman reversed-phase ODS, 4.6 × 150 m m and the mobile phase isocratic, 89% H 2 0 , 10% 2propanol, 1% glacial acetic acid, v/v/v (pH -2.5). Aliquots (10/A) of the hydroxylation reaction mixture are taken at various intervals and acidified
114
HYDROCARBONS AND RELATED COMPOUNDS
[ 18]
with 200/zl of 6 N HC1. Saturate the sample with NaC1 and extract it several times with 0.5 ml of anhydrous diethyl ether. Evaporate the ether with a stream of argon and dissolve the residual material in 100 gl of the HPLC mobile phase. After filtering, inject l0 gl of this solution directly onto the analytical column for comparison with appropriate standards. For accurate quantitations, the partition coefficients of 4HPA and HPCA into the organic phase during the extractions and their respective molar absorptivity at the HPLC monitoring wavelength must be taken into account. Procedure. The enzymatic conversation of monohydroxy aromatics to specific dihydroxy aromatics is depicted in the right half of Scheme 1. NADPH is regenerated using the standard system. Method A results in the conversion of [170]- or [1SO]hydroxyl- or carboxyl-enriched monohydroxyaromatic compound to the dihydroxy aromatic analog using unenriched 02. Method B results in the incorporation of the oxygen from 170- or 1SO-enriched 02 into the product. Method A: Synthesis of [4-170]- or [1sO]hydroxyl-enriched homoprotocatechuate. Dissolve the [4-170]- or [1SO]hydroxyl-enriched 4HPA ( - 2 0 mg) from the diazonium reaction in 5 ml of buffer and adjust the pH to 7.0 with 6 N NaOH. Add G6P (70.5 mg) and NADPH (2.5 mg) and readjust the pH to 7.0. Add catalase and G6PDH (10 units each) from 100-1000X concentrated stock solutions. Initiate the reaction by the addition of 10 units 4HPA 3-hydroxylase from a concentrated stock solution. The 02 concentration is maintained by directing a stream of compressed 02 over the stirred reaction mixture. Aliquots (10 #1) are taken every 30 min to monitor the reaction progress as discussed above. Stop the reaction when complete ( - 4 hr) by acid precipitation of the enzymes. The isolation of the [4-170]- or [1sO]hydroxyl-enriched HPCA from the reaction mixture is discussed below. Method B: Synthesis of [3-170]- or [~sO]hydroxyl-enriched homoprotocatechuate. The reaction is carded out in a glass manifold with a reaction well and a removable storage vessel equipped with a high-vacuum stopcock for recovery of the labeled 02. Add the buffer, G6P, NADPH, and 4HPA to the reaction well. Adjust the pH as above, and seal the apparatus with a rubber septum. Draw a vacuum and refill with oxygen-free argon or nitrogen several times to achieve anaerobiosis. After freezing the mixture with a dry ice-2-propanol bath, draw a full vacuum and refill with 1 atmosphere of 170- or 1SO-enriched 02. Thaw the solution and stir to ensure full 02 equilibration. In a separate vial combine the catalase, G6PDH, and 4HPA 3-hydroxylasc (10 units each) as a concentrated stock solution and make the solution anaerobic. Initiate the hydroxylation reaction by the addition of the enzymes to the reaction well through the rubber septum using a gas-tight syringe. Aliquots of the stirred solution are taken every 30 rain
[ 19]
CATECHOL 2,3-DIOXYGENASES
115
and analyzed as described above to monitor the reaction progress. When the reaction is complete, acid-precipitate the enzymes and freeze the mixture in a dry ice-2-propanol bath. The ~70- or ~80-enriched 02 is condensed in the previously evacuated storage flask with liquid N2. After isolating the storage flask, the reaction mixture is thawed and the HPCA is isolated as described below.
Isolation of[3- or 4-Iz0]- or [~80]Hydroxyl-Enriched Homoprotocatechuate. Transfer the acidified reaction mixture to a microdistillation apparatus, reduce the volume to approximately 1 ml, and saturate the solution with NaC1. Add anhydrous diethyl ether and, if an emulsion forms, centrifuge. Separate the solvent phase from the aqueous phase. The extraction is repeated several times. The combined solvent phase is dried with anhydrous MgSO4, filtered into a test tube, and evaporated under a stream of argon. Dissolve the residue in 1 ml of water containing 1.2 molar equivalents of NaOH and treat it with activated charcoal. Filter the solution into a test tube containing 5 molar equivalents of HC1, saturate with NaCI, extract with ether, dry, and evaporate as above. The product is pure, and the enrichment is essentially that of the starting labeled compound. Acknowledgements This workwas supportedby a grantfromthe NationalInstitutesof Health(GM 24689).
[ 19] C a t e c h o l 2 , 3 - D i o x y g e n a s e s f r o m P s e u d o m o n a s aeruginosa 2x
By I. A. KATAEVAand L.A. GOLOVL~.VA Cleavage of the aromatic ring is a key reaction in the oxidation of aromatic compounds. Dioxygenases capable of cleaving the aromatic ring widely occur in bacteria, but they differ in their mode of aromatic ring cleavage, specific inductions, and substrate specificity.~ Certain bacteria of the genus Pseudomonas synthesize several aromatic ring-cleaving enzymes that enable them to oxidize various aromatic compounds.2-4 For example, Pseudomonas aeruginosa 2x which grows on a wide set of aromatic subL. N. Omston, Bacteriol. Rev. 35, 87 (1971). 2 L. N. Ornston, J. Biol. Chem. 241, 3800 (1966). 3 E. A. Bamsley, J. Bacteriol. 124, 404 (1976). 4 L. N. Ornston, in "Current Topics in Cellular Regulation" (B. L. Horecker and E. R. Stadtman, eds.), Vol. 12, p. 209. Academic Press, New York, 1977.
METHODS IN ENZYMOLOGY, VOL 188
English translation copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
SYNTHESIS OF DIHYDROXY AROMATIC COMPOUNDS
107
both the C-3 and C-4 positions are also turned over, but at dramatically reduced rates (< 10% that ofgentisate). All three main functional groups of gentisate appear to be required for efficient turnover, in that little or no turnover is observed with compounds in which any one of these groups is substituted with another functionality. Additionally, monosubstituted benzoates such as salicylate, 3-hydroxybenzoate, and thiosalicylate are not turned over but are relatively good inhibitors of the enzyme. Gentisate 1,2-dioxygenase is inactivated in a time- and concentration-dependent manner by oxidants such as H202 and K3Fe(CN)6. Partial reactivation is effected by ascorbate. Acknowledgment This work was supported by a grant from the National Institutes of Health (GM 24689).
[18] S y n t h e s i s o f 170- o r 1 s O - E n r i c h e d D i h y d r o x y Aromatic Compounds B y A L L E N M . ORVILLE, M A R K R . H A R P E L , a n d JOHN D . LIPSCOMB
Specific isotopic labeling of the oxygen atoms of phenolic and catecholic compounds is of use in spectroscopic and kinetic investigations of a variety of enzymes that bind these molecules as substrates or inhibitors.t.2 A convenient and versatile synthetic procedure is depicted in Scheme 1. The position of incorporation of the isotopically enriched oxygen atom is determined by the source of the enriched oxygen (H20 or 02) and the position of substituents in the starting aromatic amine. This methodology is illustrated here with the synthesis of isotopically enriched homoprotocatechuate (3,4-dihydroxyphenylacetate, HPCA a) as an example. This procedure has appeared in a previous report. 2 These techniques have been shown to be applicable to a wide variety of phenolic and catecholic aromatic compounds (Table I).
1D. M. Arciero and J. D. Lipscomb, J. Biol. Chem. 261, 2170 (1986). 2 A. M. Orville and J. D. Lipscomb, J. Biol. Chem. 264, 8791 (1989). 3 Abbreviations used are given in Table I.
METHODS IN ENZYMOLOGY,VOL. 188
Copyright© 1990by AcademicPress,Inc. Allfightsofreproductionin any formreserved.
108
HYDROCARBONS AND RELATED COMPOUNDS
[ 18]
O"
R
I
R Diazonium Reaction
N2
R
02
OH 2
I
Hydroxylation
R
SCHEME 1
Chemical Methods
Diazonium Reaction: Synthesis 0f[170]- or [1sO]Hydroxyl-Enriched 4-Hydroxyphenylacetate Reagents 4-Aminophenylacetate (4APA), recrystallized from hot water Distilled 170- or toO-enriched water Desiccated NaNO2 Procedure. In this procedure an aromatic amine is converted to the corresponding diazonium salt and then hydrolyzed in ~0- or toO-enriched water to form the labeled phenol. The method was adapted from that of Robertson and Jacobs. 4 Formation of the diazonium salt. Add 50 mg of 4APA (1.0 molar equivalent) to a small vial containing 600/tl 170- or toO-enriched water. Cool the mixture in an ice bath with stirring and add 2.2 molar equivalents of 12 N HC1 (~ 60/tl). In a separate vial, add approximately 100 ~1170- or toO-enriched water to 1.1 molar equivalents of NaNO2 (25 mg) and cool on ice. Add the NaNO2 solution to the amine in 5-/A aliquots from a cold Hamilton syringe over 30 rain. Five minutes after the last addition of the NaNO2 add about 50 mg urea to remove the excess HONO. Hydrolysis of the diazonium salt. Heat, with continuous stirring 170- or mSO-endched water (600/A) containing 2.2 molar equivalents 12 N HC1 (-60/~1) to 90-100 ° in a two-neck pear-shaped reaction flask (10 ml) immersed in an oil bath. The vertical neck of the flask (T 14/20) is connected to a condenser, and the side neck (T10/18) is sealed with a rubber septum. Add 25-/tl aliquots of the cold diazonium salt solution through the septum with a cold syringe. Allow the evolution of N2 to subside between additions. Continue heating for 15 min after the final addition. Partial 4 G. R. Robertson and T. L. Jaeobs, in "Laboratory Practice of Organic Chemistry," 4th Ed. Macmillan, New York, 1962.
0
2, I-
N
O a~ r~
0
O
~~ ~
110
HYDROCARBONS AND RELATED COMPOUNDS
[ 18]
exchange of the carboxyl oxygens of some aromatic acids with water may occur during this step of the procedure. This exchange can be reversed as discussed in the carboxyl exchange section below. Recovery oflZO- or ~SO-enriched water. Cool the reaction to 0 °, drain the condenser, and connect the reaction flask to a short-arm vacuum distillation curve. Connect the other end of the curve to a 10-ml pearshaped collection flask. Cool both the reaction flask and the collection flask in liquid nitrogen, draw a vacuum, and then isolate the system from the vacuum line. On warming the reaction flask to 23 °, -the ~70- or nO_ enriched water is removed by distillation and is condensed in the cold collection flask. The enriched water recovered in this way must be neutralized, treated with activated charcoal, and distilled again before reuse.
Isolation of [~zO] or [1sO]hydroxyl-enriched 4-hydroxyphenylacetate. Dissolve the lyophilized hydrolysis product in 1 ml of water containing 1.2 molar equivalents of 6 N NaOH. The mixture is then treated with activated charcoal and filtered into a vial containing 5 equivalents of HC1. Saturate the filtrate with NaC1 and extract it several times with l ml of anhydrous diethyl ether. Combine the ether extracts and dry them with anhydrous MgSO 4. After filtering, evaporate the solvent under a stream of argon or by vacuum distillation. The product is pure, with an isotopic enrichment essentially the same as that of the enriched water used in the hydrolysis. This reaction proceeds readily with aniline and m- or p-amino-substituted benzoates or phenylacetates. The diazonium reaction does not produce the desired phenolic products in appreciable yields from aromatic compounds with substituents ortho to the amine.
Carboxyl Oxygen Exchange: Synthesis of [170] or [IsO]Carboxyl-Enriched Homoprotocatechuate Reagents Recrystallized HPCA Distilled ~70- or ~80-enriched water Argon or nitrogen gas passed over a column of BASF copper catalyst (Kontes, Vineland, NJ, 655960) to remove traces of contaminating 02 Procedure. The carboxylate oxygens of aromatic acids are exchanged with 170- or ~SO-enriched water via an acid-catalyzed reaction. To HPCA (50 mg) in a thick-walled hydrolysis tube (bulb v o l u m e - 1 ml) add enriched or unenriched water (550/d) and 12 N HC1 (50/11). Draw a vacuum and refill the tube with oxygen-free argon or nitrogen several times to achieve anaerobiosis. Freeze the sample, then evacuate and seal the
[18]
SYNTHESIS OF DIHYDROXY AROMATIC COMPOUNDS
II1
tube. The sealed tube is placed in an oven at 110 ° for 24 hr during which time the exchange occurs. After cooling, the sample is frozen and the tube opened. The 170- or 180-enriched water is recovered by distillation as discussed above. Mass spectral analysis of the [170] or [180]carboxylenriched HPCA shows complete exchange. Benzoate, phenylacetate, and their m- and/or p-hydroxyl-substituted derivatives yield carboxyl oxygenenriched compounds with this procedure. In the case of o-hydroxyl-substituted benzoates and phenylacetates, the incorporation is not limited exclusively to the carboxyl oxygens under the conditions described here. Conditions that promote the carboxyl oxygen exchange are briefly achieved during the hydrolysis of the diazonium salt discussed above. Undesired carboxyl oxygen enrichment can be eliminated without affecting the enrichment of the hydroxyl oxygen atom by carrying out the exchange reaction in unenriched water. Enzymatic Methods
Assay Methods Catecholic dioxygenase and aromatic hydroxylase activity assays have been described in this and earlier volumes of this series.5-s
Microbial Induction Monooxygenases of the type required for the right half of Scheme 1 are expressed by many bacteria when these organisms are cultured with the monohydroxy aromatic compound as the sole source of carbon and energy (Table I). 1,2,8-11 Several of these have been purified to homogeneity, and at least one is commercially availableJ 2 Growth of the Organism. Brevibacterium fuscum (ATCC 15933) is cultured in the medium described elsewhere in this volume5 with alteration in the carbon source as indicated below. Transfer a loop filled with cells from an agar plate containing medium plus 3 g/liter 4HBA to a 300-ml flask containing 100 ml of the same medium and carbon source. After 5 j. W. Whittaker, A. M. Orville, and J. D. Lipscomb, this volume [14]. 6 H. Fujisawa, this series, Vol. 17A, p. 526. 7 H. Kita and S. Senoh, this seres, Vol. 17A, p. 645. s M. Husain, L. M. Schopfer, and V. Massey, this series, Vol. 53, p. 543. 9 j. L. Michalover and D. W. Ribbons, Biochem. Biophys. Res. Commun. 55, 888 (1973). l°E. R. Blakley, W. Kurz, H. Halvorson, and F. J. Simpson, Can. J. Microbiol. 13, 147 (1967). 11 L-H. Wang, R. Y. Hamzah, Y. Yu, and S-C. Tu, Biochemistry 26, 1099 (1987). 124-Hydroxybenzoate 3-hydroxylase is available from Sigma Chemical Co., St. Louis, MO.
112
HYDROCARBONS AND RELATED COMPOUNDS
[18]
14 hr of growth on a rotary shaker at 30", transfer 5 ml of the culture to a 2-liter flask containing 1 liter of medium with a carbon source of 2.0 g 4HBA and 1.0 g 4HPA. After 14 hr of growth, transfer the culture to an 18-liter carboy containing 15 liters of medium with 2 g/liter 4HPA as the only carbon source. The culture is vigorously aerated ( - 1 liter/see), until the end of log phase growth ( - 24- 30 hr) with the addition of 2.0 g/liter of neutralized 4HPA after 12 hr. The cells are harvested with a water-cooledcontinuous-flow centrifuge, frozen rapidly in powdered dry ice, and stored at -80*. The yield is approximately 5 g of cells (wet weight) per liter of medium. Under these conditions, the 4HPA 3-hydroxylase and the catecholic dioxygenases protocatechuate 3,4-dioxygenase (3,4-PCD) and homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) are induced.
Purification of 4-Hydroxyphenylacetate 3-Hydroxylase Reagents MOPS buffer: 50 m M 4-morpholinopropanesulfonic acid, adjusted to pH 7.0 with 6 N NaOH Phenylmethylsulfonyl fluoride (PMSF) (Sigma, St. Louis, MO), toxic Ribonuclease and deoxyribonuclease (Sigma) Enzyme-grade ammonium sulfate (Schwarz Mann Chemical Co., Cleveland, OH) Flavin adenine dinucleotide (FAD) (Sigma) A partial purification of 4HPA 3-hydroxylase is described here. The primary objective of this preparation procedure is to separate the hydroxylase from the catecholic dioxygenases that would degrade the reaction product during the extended in vitro enzymatic hydroxylation reaction.
Step 1: Preparation of Cell-Free Extract. Frozen cells (50 g) are hammered into a fine meal and rapidly suspended in 100 ml of MOPS buffer containing 2 m M PMSF, l0 m M EDTA, 1 m M 2-mercaptoethanol (2-ME), 10/~M FAD, and 2 mg each ribonuclease and deoxyribonuclease. Sonicate the cells for 45 min at 80% power with a Branson Model 350 sonicator equipped with a 3/4-inch fiat tip. The temperature is maintained at 5-10*with a 2-propanol-dry ice bath. Remove the cell debris by centrifugation for 30 min at 22,000 g and 4". Step 2: Ammonium Sulfate Fractionation. The cell-free extract is made 55% saturated in ammonium sulfate, the pH is adjusted to 7.0 with 6 N NaOH, and the solution is centrifuged as above. All of the 3,4-PCD and approximately half of the 2,3-HPCD is found in the pellet. Bring the supernatant to 70% saturation in ammonium sulfate (pH 7.0) to precipitate the 4HPA 3-hydroxylase and residual 2,3-HPCD and centrifuge.
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SYNTHESIS OF DIHYDROXY AROMATICCOMPOUNDS
113
Step 3: Ion-Exchange Chromatography. Dissolve the pellet in 10 ml of MOPS buffer containing 2 m M PMSF and centrifuge at 22,000 g and 4 ° for 15 min to remove any insoluble protein. Dilute the supernatant to 200 ml and load it onto a column of Whatman DE-52 (3 × 18 cm) equilibrated with buffer. After washing with 75 m M ammonium sulfate in buffer, elute the residual 2,3-HPCD with a linear gradient (100 and 100 ml) from 75 to 150 m M ammonium sulfate in buffer. Elute the 4HPA 3-hydroxylase activity with a linear gradient (250 and 250 ml) from 150 to 300 m M ammonium sulfate in buffer. Fractions with more than 0.5 units/ ml hydroxylase activity are pooled and reactivated (-400%) by incubation with 1 m M 2-ME and 10/tM FAD for 20 min. Step 4: Concentration and Storage. Precipitate the reactivated hydroxylase by bringing the solution to 70% saturation in ammonium sulfate (pH 7.0). After centrifugation, redissolve the pellet in 3 ml of MOPS buffer containing 1 m M 2-ME and 10/zM FAD. Typically this preparation has approximately 70 units/ml and a specific activity of about 20 units/mg protein. The enzyme is frozen with liquid N2 and can be stored at 77 K for several months without loss of activity. Synthesis of [~70]- or [1sO]Hydroxyl-Enriched Homoprotocatechuate Reagents Buffer, 0.25 M MOPS, pH 7.0, containing 1 m M 2-ME and 10 FAD Glucose 6-phosphate (G6P), 50 m M 4HPA, 25 mM, unenriched or [170]- or [180]hydroxyl-enriched NADPH, 0.6 m M 170- or 1SO-enriched or unenriched 02 Glucose-6-phosphate dehydrogenase (G6PDH) (Sigma) 4HPA 3-hydroxylase Monitoring Hydroxylase Reaction Progress. The extended in vitro hydroxylase reaction progress can be followed in most cases by monitoring the loss of the monohydroxyaromatic substrate and/or increase in dihydroxyaromatic product with a UV spectrophotometer. However, the electronic spectra of 4HPA (2m~ 278 nm, ~ 1400 M -1 cm -1) and HPCA ( 2 ~ 282 nm, e 2500 M -~ cm -t) do not permit simple independent quantitations in a complex mixture. Determination of the product concentration using HPLC is a suitable alternative. HPLC Determinations. The column is a Beckman reversed-phase ODS, 4.6 × 150 m m and the mobile phase isocratic, 89% H 2 0 , 10% 2propanol, 1% glacial acetic acid, v/v/v (pH -2.5). Aliquots (10/A) of the hydroxylation reaction mixture are taken at various intervals and acidified
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HYDROCARBONS AND RELATED COMPOUNDS
[ 18]
with 200/zl of 6 N HC1. Saturate the sample with NaC1 and extract it several times with 0.5 ml of anhydrous diethyl ether. Evaporate the ether with a stream of argon and dissolve the residual material in 100 gl of the HPLC mobile phase. After filtering, inject l0 gl of this solution directly onto the analytical column for comparison with appropriate standards. For accurate quantitations, the partition coefficients of 4HPA and HPCA into the organic phase during the extractions and their respective molar absorptivity at the HPLC monitoring wavelength must be taken into account. Procedure. The enzymatic conversation of monohydroxy aromatics to specific dihydroxy aromatics is depicted in the right half of Scheme 1. NADPH is regenerated using the standard system. Method A results in the conversion of [170]- or [1SO]hydroxyl- or carboxyl-enriched monohydroxyaromatic compound to the dihydroxy aromatic analog using unenriched 02. Method B results in the incorporation of the oxygen from 170- or 1SO-enriched 02 into the product. Method A: Synthesis of [4-170]- or [1sO]hydroxyl-enriched homoprotocatechuate. Dissolve the [4-170]- or [1SO]hydroxyl-enriched 4HPA ( - 2 0 mg) from the diazonium reaction in 5 ml of buffer and adjust the pH to 7.0 with 6 N NaOH. Add G6P (70.5 mg) and NADPH (2.5 mg) and readjust the pH to 7.0. Add catalase and G6PDH (10 units each) from 100-1000X concentrated stock solutions. Initiate the reaction by the addition of 10 units 4HPA 3-hydroxylase from a concentrated stock solution. The 02 concentration is maintained by directing a stream of compressed 02 over the stirred reaction mixture. Aliquots (10 #1) are taken every 30 min to monitor the reaction progress as discussed above. Stop the reaction when complete ( - 4 hr) by acid precipitation of the enzymes. The isolation of the [4-170]- or [1sO]hydroxyl-enriched HPCA from the reaction mixture is discussed below. Method B: Synthesis of [3-170]- or [~sO]hydroxyl-enriched homoprotocatechuate. The reaction is carded out in a glass manifold with a reaction well and a removable storage vessel equipped with a high-vacuum stopcock for recovery of the labeled 02. Add the buffer, G6P, NADPH, and 4HPA to the reaction well. Adjust the pH as above, and seal the apparatus with a rubber septum. Draw a vacuum and refill with oxygen-free argon or nitrogen several times to achieve anaerobiosis. After freezing the mixture with a dry ice-2-propanol bath, draw a full vacuum and refill with 1 atmosphere of 170- or 1SO-enriched 02. Thaw the solution and stir to ensure full 02 equilibration. In a separate vial combine the catalase, G6PDH, and 4HPA 3-hydroxylasc (10 units each) as a concentrated stock solution and make the solution anaerobic. Initiate the hydroxylation reaction by the addition of the enzymes to the reaction well through the rubber septum using a gas-tight syringe. Aliquots of the stirred solution are taken every 30 rain
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CATECHOL 2,3-DIOXYGENASES
115
and analyzed as described above to monitor the reaction progress. When the reaction is complete, acid-precipitate the enzymes and freeze the mixture in a dry ice-2-propanol bath. The ~70- or ~80-enriched 02 is condensed in the previously evacuated storage flask with liquid N2. After isolating the storage flask, the reaction mixture is thawed and the HPCA is isolated as described below.
Isolation of[3- or 4-Iz0]- or [~80]Hydroxyl-Enriched Homoprotocatechuate. Transfer the acidified reaction mixture to a microdistillation apparatus, reduce the volume to approximately 1 ml, and saturate the solution with NaC1. Add anhydrous diethyl ether and, if an emulsion forms, centrifuge. Separate the solvent phase from the aqueous phase. The extraction is repeated several times. The combined solvent phase is dried with anhydrous MgSO4, filtered into a test tube, and evaporated under a stream of argon. Dissolve the residue in 1 ml of water containing 1.2 molar equivalents of NaOH and treat it with activated charcoal. Filter the solution into a test tube containing 5 molar equivalents of HC1, saturate with NaCI, extract with ether, dry, and evaporate as above. The product is pure, and the enrichment is essentially that of the starting labeled compound. Acknowledgements This workwas supportedby a grantfromthe NationalInstitutesof Health(GM 24689).
[ 19] C a t e c h o l 2 , 3 - D i o x y g e n a s e s f r o m P s e u d o m o n a s aeruginosa 2x
By I. A. KATAEVAand L.A. GOLOVL~.VA Cleavage of the aromatic ring is a key reaction in the oxidation of aromatic compounds. Dioxygenases capable of cleaving the aromatic ring widely occur in bacteria, but they differ in their mode of aromatic ring cleavage, specific inductions, and substrate specificity.~ Certain bacteria of the genus Pseudomonas synthesize several aromatic ring-cleaving enzymes that enable them to oxidize various aromatic compounds.2-4 For example, Pseudomonas aeruginosa 2x which grows on a wide set of aromatic subL. N. Omston, Bacteriol. Rev. 35, 87 (1971). 2 L. N. Ornston, J. Biol. Chem. 241, 3800 (1966). 3 E. A. Bamsley, J. Bacteriol. 124, 404 (1976). 4 L. N. Ornston, in "Current Topics in Cellular Regulation" (B. L. Horecker and E. R. Stadtman, eds.), Vol. 12, p. 209. Academic Press, New York, 1977.
METHODS IN ENZYMOLOGY, VOL 188
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