THEJOURNAL OF Bmmcmnr. CHEMISTRY Vol. 245, No. 21, Issue of November 10, pp. 5857-5864, 1970

Ptininted in U.S.A.

The Metabolism of Cyclopropanecarboxylic Acid* (Received for publication, June 24, 1970)

JULUN G. SCHILLER AND ALBERT E. CHUNG

From the Department of Biochemistry and Nutrition, Graduate School of Public Health and Department of Bio- chemistry, Faculty of Arts and Scierzces, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

SUMMARY

A pathway for the degradation of the cyclopropane ring in cyclopropanecarboxylic acid has been indicated. The fungus Fusarium oxysporum Schlectendahl converts cyclopropane- carboxylic acid to y-hydroxybutyric acid through a derivative of cyclopropanecarboxylic acid. This derivative of cyclo- propanecarboxylic acid, but not free cyclopropanecarboxylic acid, can be converted to y-hydroxybutyric acid by cell-free extracts of the microorganism. The enzymatic activity of the extract is labile and markedly dependent on pH.

The cyclopropane fatty acids cis-11,12-methyleneoctadeca- noate and &s-Q, IO-methylenehexadecanoate, occur in the phos- pholipids of many bacteria (2). The biosynthesis of the cyclo- propane ring in these compounds has been studied in detail (3-10). In contrast to these detailed studies on the biosynthesis of the cyclopropane ring very little is known about its further metabolism. Wood and Reiser (11) and Chung (12) have indi- cated that the cyclopropane ring is not metabolized by whole rats or rat liver mitochondria. Recently, Kaneshiro and Thomas (13) have indicated that if Agrobacterium tumefaciens cells are grown under special conditions, the cyclopropane fatty acids in these microorganisms may serve as precursors for the correspond- ing methyl branched chain fatty acids. The experiments de- scribed in this communication were undertaken to determine whether the cyclopropane ring was biodegradable and to estab- lish the pathway for degradation. For these goals the metab- olism of cyclopropanecarboxylic acid by Fusarium oxysporum Schlectendahl was studied.

MATERIALS

Cyclopropyl bromide, cyclopropanecarboxylic acid, 2,5-di- phenyloxazole, y-butyrolactone, and y-hydroxybutyric acid were purchased from Aldrich. Ba14C03 and sodium y-hydroxybutyr- ate-1-14C were supplied by Calbiochem and Schwarz BioResearch, respectively. Naphthalene (recrystallized from alcohol) was

* This work was supported by Grant GB-7499 from the National Science Foundation and Grant AI-08273 from the National Insti- tutes of Health. The data presented were taken from a disserta- tion submitted by J. G. Schiller to the Department of Biochemis- try and Nutrition, Graduate School of Public Health, University of Pittsburgh in partial fulfillment of the requirements for the degree of Doctor of Philosophy (1).

purchased from Eastman and p-dioxane from J. T. Baker Chemi- cal Company, Phillipsburg, N. J. Sephadex G-10 was obtained from Pharmacia and Drierite was from the W. H. Hammond Drierite Company, Xenid, Ohio. The diethyleneglycol suc- &ate polyester (15% w/w) coated onto Anakrom lOO/llO was a gift from Dr. 0. K. Reiss. All other chemicals were of re- agent grade.

METHODS

Isolation, Identification, and Growth of F. oxysporum Schlec- tendah&-The fungus F. oxysporum Schlectendahl was isolated as a contaminant during an experiment designed to adapt A. tume- faciens tt 10 to grow on cyclopropanecarboxylic acid. The fun- gus was capable of utilizing cyclopropanecarboxylic acid as its sole source of carbon and energy, whereas the A. tumejaciens could not grow on this compound. The identity of the fungus was established by the Centraal Bureau Voor Schimmelcultures, Baarn, The Netherlands. A culture of F. oxysporum (ATCC No. 659) obtained from the American Type Culture Collection was also capable of growing on cyclopropanecarboxylic acid. The experiments described here, however, were carried out with the microorganism isolated in our laboratory. The microorga- nism was grown in the medium described by Starr (14) for A. tumefaciens except that glucose was replaced by 0.5 g of cyclo- propanecarboxylic acid/100 ml of growth medium as the only source of carbon. Stock cultures were maintained at 4” on 2% w/w agar slopes prepared with the growth medium.

Cells were obtained for experimentation by transferring a piece of agar slant containing the microorganism to 250 ml of growth medium contained in a 500-ml Erlenmeyer flask. The flask was kept at 30” and agitated at a setting of six in a New Brunswick Gyrotory Shaker (New Brunswick Scientific Com- pany, Inc., New Brunswick, New Jersey) for 5 to 6 days. At this time, a loo-ml aliquot of cell suspension was used to inoculate 1 liter of fresh growth medium contained in a 2-liter Erlenmeyer flask. After 48 to 72 hours of further growth under the above conditions, the cells were harvested by filtration through two lay- ers of nylon cloth supported by a Buchner funnel. The cells, while still contained in the nylon cloth, were washed free of adhering growth medium.

Preparation of Cell-free Extracts of F. oxysporum-The follow- ing protocol was employed to prepare a cell-free extract from 12 g of Fusarium cells. Different quantities of extract may be pre- pared by using proportionately larger or smaller vessels.

The fungi obtained from 6 liters of growth medium were placed in a 20-cm Buchner funnel which contained a piece of Whatman

5857

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FIG. 1. Mass spectrum of cyclopropanecarboxylic acid. An aliquot of a benzene solution containing either standard cyclo- propanecarboxylic acid or synthetic cyclopropanecarboxylic acid-l-% was injected onto a glass column, 6 feet by + inch, packed with 1570 diethyleneglycol succinate coated onto Ana- khrom (lOO/llO mesh) maintained at 140”. The column was operated in an LKB 9000 combined gas-liquid chromatograph and mass spectrometer. Spectra were recorded at 70 electron volts. The electron current and accelerating voltage were, respectively, 60 pa and 3.5 kvolts. The molecular separator and ion source temperatures were, respectively, 250 and 270”. After the spectra were corrected for background, bar graphs were constructed by assigning the ions with mass to charge ratios (m/e) of 39 the relative intensity of 100%.

No. 1 filter paper. While on the filter, the cells were covered with 300 ml of water at 2”. The water was then drawn through the cellular mat by reducing the pressure under the filter. Next, the mat was peeled from the filter, weighed, and placed in a loo-ml glass beaker. Here, the mat was molded to form a I- to 3-mm layer of fungi that covered the bottom of the beaker and one- quarter to one-third of its wall area. The contents of the beaker were then frozen by immersing the beaker in a bath composed of dry ice and acetone for 5 min. Four volumes of 0.1 M potassium phosphate buffer at 0” and the appropriate pH were added to the beaker which was surrounded by ice. The cells were allowed to thaw at 0” and the resulting thick suspension, containing as few clumps as possible, was subjected to sonic disruption for 1 min with a Heat Systems Sonifier Cell Disruptor (Heat Systems Inc., Great Neck, New York) operating at 70% of maximum out- put,. The resulting smooth mixture was then transferred to a Rosett cell (Heat Systems Inc.) whose total capacity was 275 ml. All fungal clumps were excluded to avoid clogging the cell’s cooling arms. The cell was then immersed in a stirred ice-water bath and sonic disruption begun as above. When it was ascertained that the fungal suspension was freely cir- culating throughout the cell the ice-water bath was saturated with sodium chloride. In this way, the fungal suspension was maintained at 5-6” for the remaining 10 min of sonic disruption. After this time an additional volume of 0.1 M potassium phosphate buffer was added to the mixture which was then centrifuged for 15 min in the cold at 35,000 x g in a Sorvall RCkB refrigerated centrifuge equipped with an SS-1 rotor. The supernatant from this centrifugation was taken as the cell-free system. The pH of the supernatants was determined with a Beckman Expandomatic pH meter and protein concentrations were measured by the method of Gornall, Bardawill, and David (15) with bovine se- rum albumin as a standard.

Synthesis and Purity of Cyclopropanecarboxylic Acid-l-W- Cyclopropanecarboxylic acid-l-14C was synthesized from cyclo-

propyl bromide according to Renk et al. (16) with the substitu- tion of Ba14C03 for Ba13C03. The product taken for analysis and used for all metabolic studies distilled at 117-119”/82 mm of mercury (reported (17) at 117-118”/75 mm of mercury) and had a specific radioactivity of 0.25 mCi per mmole. The mass spec- trum (Fig. 1) of the synthetic material was indistinguishable from the spectrum of cyclopropanecarboxylic acid obtained from Aldrich. Gas-liquid chromatography of synthetic material showed only one peak. The chromatography was performed isothermally at 10” intervals from 130-190” on a 15% diethylene- glycol succinate column operated as described in the legend to Fig. 3. Whenthecolumneffluent was monitored for radioactivity by employing a stream splitter, the radioactivity was found to be coincident with the chromatographic peak. As a final test for radiopurity, a sample of synthetic material was chromatographed on a column, 1.9 x 114 cm, of Sephadex G-10 as described in this section. Only one radioactive peak appeared and its retention volume was 210 ml.

Incubation of F. oxysporum Cells with Cyclopropanecarboxylic Acid-lJ4C and Isolation of Reaction ProductsThe cells from 4 liters of growth medium, concentrated to a thick suspension (50 to 80 ml) by filtration, were washed with 1 liter of growth medium at pH 7 and 30” that contained no carbon source. After the excess wash medium was removed by filtration the cells were scraped together to form a loose clump which was quickly trans- ferred to a 500-ml cylinder. In order to create a smooth suspen- sion of cells the cylinder, which contained 200 ml of growth me- dium as above, was inverted several times. The suspension was then poured into a 750-ml Erlenmeyer flask containing 60 $Zi of cyclopropanecarboxylic acid-l-X! (0.25 mCi per mmole) in 10 ml of growth medium at pH 7 that contained no other source of car- bon. The mixture was rapidly swirled for 10 set and then poured into a 20-cm Buchner funnel containing a circle of moistened Whatman No. 1 filter paper. The suspending fluid was rapidly removed by filtration and the remaining cellular mat immedi- ately washed with water at 2”. The wash water was also re- moved by filtration. Several more washes were performed with a total volume of 1 liter of water. Immediately after the last wash, the filter paper, with its adhering cellular mat,, was torn into eight pieces. These pieces were quickly dropped into 200 ml of acetone at - 17” contained in a Waring Blendor. After 80 set of blending at high speed, the slurry was filtered under suc- tion and the residual mat, composed of paper and cell debris, stirred with 200 ml of water at 4” for 20 min. After this time the resulting suspension was centrifuged at 105,000 x g in a Spinco No. 30 rotor for 30 min. The sediment was discarded and the supernatant saved. The acetone filtrate, obtained previously, was concentrated under reduced pressure at 44” to remove the acetone. The residue, usually 4 to 7 ml, was free of acetone. Water (10 ml) was added to this residue and the suspension cen- trifuged as previously described. The 105,000 x g supernatant solutions obtained from the acetone powder extract and acetone extract, were then pooled and concentrated under reduced pres- sure at 44” to 20 ml. This concentrate, contained in a 50-ml Erlenmeyer flask, was heated in a boiling water bath for 2 min, chilled, and the precipitate removed by centrifugation at 35,000 x g for 10 min in a Sorvall RC2-B refrigerated centrifuge equipped with an SS-1 rotor. The supernatant was concen- trated to 3 ml as described and chilled to 4” in preparation for Sephadex G-10 chromatography.

Sephadex G-10 Chromatography and Liquid Scintillation Spec-

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r

CPI CP f 'CA

1

I

0 ,,J 0 I) 140 160 180 200 220

Effluent Volumecml)

FIG. 2. Sephadex G-10 chromatography of the products ob- tained from an incubation of Fusarium cells with cyclopropane- carboxylic acid-l-W. The column effluent was monitored for radioactivity by assaying O.l-ml aliquots of each fraction as described under “Methods.” CPCA, cyclopropanecarboxylic acid; GHB, -y-hydroxybutyric acid.

trometry-Four columns, 1.9 x 114 cm, of Sephadex G-10,q euili- brated with 0.05 M potassium phosphate buffer, pH 7.0, were packed at room temperature, and then transferred to a cold room which was maintained at 2-4”. All subsequent Sephadex G-10 analyses were performed within this temperature range utilizing 0.05 M potassium phosphate buffer, pH 7.0, as the eluting solvent. The column flow rate was maintained between 10 and 15 ml per hour during an analysis, the sample size never exceeding 3.5 ml. By maintaining these limits for column flow rate and sample size, no variation exceeding ZIZ 5% was ever observed in either the relative or absolute retention volumes for standard substances. To determine the void volume of these columns, samples of Blue Dextran 2000 were applied to the columns and found to emerge in a lo-ml volume after 120 ml of effluent was collected.

Recoveries of radioactivity from the column ranged from 75 to 95% regardless of the substance chromatographed. During all analyses, fractions were collected at 20-mm intervals. Ah- quots of up to 2.0 ml of each fraction were assayed for radioac- tivity by placing them in 10 ml of Beckman Cocktail D (18). A Beckman LS-100 liquid scintillation spectrometer was employed and samples of carbon 14 counted at an efficiency of 73% for 5- or lo-min periods. No quenching was observed as determined by internal and external standard measurements.

RESULTS

Metabolic Products of Cyclopropanecarboxylic Acid-l-raC--The radioactive products that resulted from a lo-set incubation of Fusarium cells with cyclopropanecarboxylic acid-l-W (see “Methods”) were separated on a column of Sephadex G-10. The elution profile of radioactivity is shown in Fig. 2. These

Minutes FIG. 3. Gas-liquid chromatography of radioactive r-butyrolac-

tone. To obtain this tracing, a coiled steel column, + inch by q feet, packed with 15’% diethyleneglycol succinate polyester coated onto Anakhrom lOO/llO, was operated isothermally at 100” in a Perkin Elmer 906 gas chromatograph equipped with a hydrogen flame ionization detector. The temperature of both the injection port and manifold was 200” and the carrier gas (helium) flowed at 60 to 70 ml per min. The gaseous column effluent stream was divided such that while 2% of the stream passed through the detector, the remaining 98% was passed into a glass spiral im- mersed in a Dry Ice-acetone bath. The portion of the effluent trapped in this manner was washed from the spiral with scintilla- tion fluid which was placed in a vial to be assayed for radioac- tivity. A Beckman LS-100 liquid scintillation spectrometer was employed for this purpose. Samples were counted to a percentage error of ~2% and no quenching was observed as determined by use of an external standard.

100

r- a.

7s -

25 -

JL- b.

0

Y ‘, I 50 60 70 80 SO

ve FIG. 4. Mass spectra of (a) “Unknown” and (b) r-butyrolac-

tone. An aliquot of the benzene solution containing either r-butyrolactone or “Unknown” was subjected to combined gas- liquid chromatography and mass spectrometry under conditions identical with those described in the legend to Fig. 1. After the spectra were corrected for background, bar graphs were constructed by assigning the ions with mass to charge ratios (m/e) of 42 the relative intensity of 100%.

products usually contained from 0.25 to 0.60 ‘% of the radioactiv- ity with which the incubation was begun. The remainder of the radioactivity was recovered as cyclopropanecarboxylic acid-l- 1% in the incubation medium. When the duration of the incu-

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"0 N120 140 160 180 200 220

Effluent Volume (ml)

FIG. 5. Sephadex G-10 chromatography of identical aliquots of a solution of cyclopropanecarboxylic acid-X (a) before and (b) af- terbasic hydrolysis. Thesolution of cyclopropanecarboxylic acid- X t.aken for hydrolysis was made to 5% with respect to KOH (w/v) and heated at 100” for 2 hours. After this time the pH of the solution was adjusted to 7 with 12 N HCl and the neutral solution applied to the Sephadex G-10 column. The column was eluted, fractions of 4 ml collected and 2.0-ml aliquots of each fraction assayed for radioactivity as described under “Methods.” The specific radioactivity of the liberated cyclopropanecarboxylic acid was found to be approximately 0.016 mCi per mmole and was determined by gas-liquid chromatography and liquid scintillation spectrometry as also described under “Methods”.

bation was increased, the radioactive peaks became much less sharply defined. Ample radioactive material was obtained for analysis by pooling corresponding fractions from independent experiments.

The radioactive material in the peak labeled yclopropunecar- boxy& acid (Fig. 2) was identified as cyclopropanecarboxylic acid by gas-liquid chromatography and mass spectrometry as pre- viously described for cyclopropanecarboxylic acid-lJ4C. In preparation for gas-liquid chromatography, the appropriate frac- tions from the Sephadex G-10 column were acidified with hydro- chloric acid. The acid solution was extracted four times in suc- cession with twice its volume of diethyl ether. In this way 95% of the radioactivity initially present in the aqueous phase was found in the pooled ether extracts. These extracts were dried with Drierite and the ether evaporated under reduced pressure. The residue was dissolved in benzene for gas-liquid chromato- graphic analysis. Since cyclopropanecarboxylic acid is volatile, the benzene solution contained only 50% of the radioactivity found initially in the pooled ether extracts.

The radioactive material constituting the peak labeled GHB in Fig. 2 was identified as y-hydroxybutyrate. A portion of the Sephadex G-10 effluent containing this material was acidified with hydrochloric acid to a pH less than 1 and continuously ex- tracted with diethyl ether for 15 hours. After this time 90% of the radioactivity was present in the ether phase. The ether ex-

TABLE I Metabolism of cyclopropanecarboxylie acid and

cyclopropanecarboxylic acid-X by cell-free extracts of F. oxysporum

The cell-free extract employed for these experiments was ob- tained as described under “Methods.” The buffer used to sus- pend the cells for sonic disruption was 0.1 M potassium phosphate at pH 6.7 and the protein concentration of the cell-free extract was 7.5 mg per ml. In Experiment 1 the incubation mixture contained 2.5 ml of cell-free extract and 0.03 PCi of cyclopropane- carboxylic acid-l-W (specific activity 0.25 pCi per pmole) in 5 pl of water. In Experiment 2 the incubation mixture contained 7.2 ml of cell-free extract and 0.005 pCi of radiopure 14C-labeled cyclopropanecarboxylic acid-X in 0.7 ml of 0.05 M potassium phos- phate buffer, pH 7.0. The reactions were initiated by addition of substrates and the mixtures incubated in 254 Erlenmeyer flasks in a Dubnoff metabolic shaker at 30”. The reactions were termi- nated at the end of 30 min by heating the reaction mixtures for 2 min in a boiling water bath. The resulting mixture was quickly chilled and centrifuged for 10 m.in at 23,000 X g in a Sorvall RCP-B refrigerated centrifuge equipped with an SS-1 rotor. Each super- natant solution was then concentrated under reduced pressure at 42” and applied to a column, 1.9 X 114 cm, of Sephadex G-10. The column was eluted, 4-ml fractions collected and 2.0 ml of each fraction assayed for radioactivity as described under “Methods.” After determining the amount of radioactivity recovered from each Sephadex column, the percentage of this total represented by cyclopropanecarboxylic acid, cyclopropanecarboxylic acid-X, r-hydroxybutyric acid, or [?I was calculated and the results shown in this table. The over-all recovery of radioactivity in the two experiments were 75 and 85yo, respectively. In the absence of any cell-free extract, cyclopropanecarboxylic acid-X under simi- lar conditions of incubation was recovered unchanged.

I Total radioactivity recovered

Experiment 1 cyclopropanecarbox-

ylic acid. . , . Experiment 2

cyclopropanecarbox- ylic acid-X. .

Cyclopro- pant?c+r- b;zp

%

99.9

53.5

<O.Ol <O.Ol

35.0 2.0

tract was dried with Drierite and the ether evaporated under re- duced pressure. Evaporation accounted for a 50% loss in radio- activity during this procedure. The residue was then dissolved in benzene in preparation for gas-liquid chromatographic analy- sis.

Chromatography of aliquots of the benzene solution, per- formed on the 15% diethyleneglycol succinate polyester column, revealed two peaks that were well separated in the range from 100-170” as shown in Fig. 3. The peak with longer retention time was inseparable from authentic y-butyrolactone at 10” inter- vals from 100-170”. The stream splitter (see legend to Fig. 3) allowed the column effluent to be monitored for radioactivity, showing that approximately 90% of the recoverable radioactivity (93% in this instance) was associated with the peak area corre- sponding to y-butyrolactone. Combined gas-liquid chromatog- raphy and mass spectrometry confirmed the identification. The

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TABLE II Metabolism of r-hydroxybutyric acid-l-Y! by

cell-free extracts of F. oxysporum The complete reaction mixture contained 6 ml of crude extract

containing 57 mg of protein; 700 pmoles of potassium phosphate buffer, pH 7.6; 1.95 pmoles of y-hydroxybutyric acid-l-l% con- taining 5.5 X 1W3&i of radioactivity, and water to a total volume of 7 ml. The control vessel contained the same components except that prior to addition of r-hydroxybutyric acid-l-W the extract was boiled for 2 min. In Experiment 2, 71 @moles of unlabeled r-hydroxybutyrate, contained in 1.0 ml of water at pH 7.6, were added to the complete system. The reaction mixtures were incubated at 30”, with shaking, for 90 min. At the end of the incubation the reaction was terminated by boiling and the samples prepared for analysis as described in Table I.

System

- I

Experiment 1 Complete. . . . . . . . . . .

Experiment 2 Complete + r-hydroxybutyric acid. .

Experiment 3 Control. . . . . . . . . . . . . . . . . . . . . . . . . .

Total recovered radioactivity

y-hydroxy- butyric acid

% %

72.0 28.0

97.5 2.5

99.5 0.5

VI

mass spectra for authentic y-butyrolactone and the “Unknown” are shown in Fig. 4.

The reaction product isolated from the Sephadex G-10 column was not the lactone but y-hydroxybutyrate. This conclusion is based upon the observations that (a) the product was not ex- tractable from the aqueous solution by benzene unless it was first heated in 1 N HCl at 100” for 30 min, these conditions convert the free acid to the la&one which becomes extractable with benzene, (b) after heating in acid and extraction with benzene this product cochromatographed with authentic y-butyrolactone on 15% diethyleneglycol succinate polyester and yielded a mass spectrum identical withauthentic y-butyrolactone, (c) rechromatography of the isolated radioactive y-butyrolactone on Sephadex G-10 yielded a single sharp radioactive band with a retention volume characteristic for y-butyrolactone, (d) the retention volume on Sephadex G-10 of the original product was identical with that of authentic y-hydroxybutyric acid.

The radioactive peak labeled CPC~-X was isolated free from contaminating radioactive impurities by repeated chromatog- raphy of the peak fractions on Sephadex G-10. The elution profile of the final product on Sephadex G-10 is shown in Fig. 5~. Further support for its radiopurity was obtained by thin layer chromatography. Chromatography of this radioactive material on cellulose plates developed in chloroform-methanol-water (15: 75: 12) or 1-butanol-acetic acid-water (4: 1: 1) showed that the radioactive material had an Rp of 0.8 in the former system and an RF of 0.7 in the latter. An Rp of 0.07 was obtained when the material was chromatographed on a silica gel plate in the f&t solvent system. The plates were analyzed for radioactivity by mixing l-cm2 areas of the supporting medium with 2.0 ml of water and then adding 10 ml of Beckman Cocktail D (18). No more than one band of radioactivity was observed on any of the three thin layer plates. The radioactive cyclopropanecarboxylic acid-X was shown to be a derivative of cyclopropanecarboxylic

( ;::jc7;> : k? -*

O0 6.1 6.5 6.9 1.3 7.1

pfl of Incubation

FIG. 6. The metabolism of cyclopropanecarboxylic acid-X by the cell-free system as a function of pH. Five 1.8-g portions of cells were suspended for sonic disruption (see “Methods”) in 0.1 M potassium phosphate buffer solutions at pH 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The pH of each cell-free extract ob- tained was, in the same order: 6.15, 6.52, 6.90,7.35, and 7.55. The range of protein concentrations was from 8.25 to 8.5 mg per ml with the exception of the supernatant solution at pH 6.15. This supernatant solution had a protein concentration of 6.75 mg per ml. The substrate for the incubations was a solution of cyclo- propanecarboxylic acid-X (see Fig. 5a for G-10 profile) containing 11,000 cpm per ml. A 0.7-ml aliquot of this solution was added to 7.0-ml aliquots of the various cell-free extracts. Each incuba- tion was conducted at 30” in a 25-ml Erlenmeyer flask for 90 min. The flasks were agitated in a Dubnoff Metabolic Shaker. The incubations were terminated and samples prepared for and ana- lyzed by Sephadex G-10 chromatography as described in the legend to Table I. After determining the amount of radioactivity recovered from each G-10 column, the percentage of this total represented by r-hydroxybutyric acid, cyclopropanecarboxylic acid, cyclopropanecarboxylic acid-X, or [?] was calculated. The results of these calculations are shown. The over-all recoveries of radioactivity ranged from 67 to 77%. CPCA, cyclopropane- carboxylic acid; GHB, r-hydroxybutyric acid.

acid. Mild acidic or basic hydrolysis of the radioactive material yielded quantitatively an ether-extractable material which chro- matographed on Sephadex G-10 with a retention volume iden- tical with that of cyclopropanecarboxylic acid. In Fig. 5b the elution profile for the product obtained after basic hydrolysis of cyclopropanecarboxylic acid-X is shown. Acid hydrolysis yielded an identical product. The identity of the released cyclo- propanecarboxylic acid was confirmed by gas-liquid chromatog- raphy and mass spectrometry as previously described. The na- ture of the group attached to cyclopropanecarboxylic acid is unknown.

Metabolic Relationships between C@opropanecarboxylic Acid, Cyclopropanecarboxylic Acid-X, and y-Hydroxybutyric Acid- The metabolic relationships between the radioactive compounds obtained in the whole-cell experiments were investigated with cell-free extracts of the microorganism. Table I summarizes the results obtained when cell-free extracts of the microorganism were incubated with either cyclopropanecarboxylic acid-l-14C or cyclopropanecarboxylic acid-X-14C and the products examined by Sephadex chromatography. The results clearly show that although the cell-free extract cannot metabolize cyclopropane- carboxylic acid the conversion of cyclopropanecarboxylic acid-X to cyclopropanecarboxylic acid, y-hydroxybutyric acid, and other metabolic products designated [?] occurs readily. The identity of these incubation products was established as pre- viously described. These observations support a pathway in which cyclopropanecarboxylic acid is first activated to cyclo-

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FIG. 7. The time course of the metabolism of cyclopropanecar- boxylic acid-X by the cell-free system. Cell-free extract (44 ml) at pH 6.5 and with a protein concentration of 8.7 mg per ml were prepared as described under “Met,hods.” The 0.1 M potassium phosphate buffer in which the cells were suspended for sonic dis- ruption was at pH 6.7. The extract was brought to 30” and the incubation begun by the addition of 4.9 ml of the cyclopropanecar- boxylic acid-X solution described in the legend to Fig. 6. The incubation vessel, a 250-ml Erlenmeyer flask, was maintained at 30” in a New Brunswick Gyrotary Shaker at a setting of SIX. At the times indicated, 6.0-ml aliquots of the incubation mixture were placed in tubes which were then immersed in a bath of boiling water for 2 min. The tubes were quickly chilled and samples prepared for and analyzed by Sephadex G-10 chromatography as described in the legend to Table I. After determining the total amount of radioactivity recoverable from each column, the ap- propriate percentage of this total was assigned to cyclopropane- carboxylic acid-X, cgclopropanecarboxylic acid, y-hvdroxvbutvric acid, and [?I. The- over-all recovery-of radioactivity from-the columns ranged from 68 to 80%. GHB, r-hydroxybutyric acid; CPCA, cyclopropanecarboxylic acid.

propanecarboxylic acid-X and the cyclopropanecarboxylic acid-X is converted to y-hydroxybutyric acid. The y-hydroxy- butyric acid is then converted to the terminal reaction products [?I. Further support for this pathway was obtained by incubat- ing y-hydroxybutyric acid with the cell-free extract. In this experiment the only radioactive compounds isolated were y-hy- droxybutyric acid and the terminal products [?I. The y-hy- droxybutyric acid was not converted to either cyclopropanecar- boxylic acid or cyclopropanecarboxylic acid-X. Furthermore, the addition of unlabeled y-hydroxybutyric acid decreased the conversion of the labeled compound to labeled products. These data are summarized in Table II. Thin layer chromatography indicated that [?] consists of several components which were not further characterized. The conversion of cyclopropanecarbox- ylic-X to cyclopropanecarboxylic acid by cell-free extracts was al- ways observed during these experiments. This conversion is probably caused by a specific cyclopropanecarboxylic-X “hydro- lase” which is distinct from the ring-breaking activity. In fact, this hydrolase activity can be completely separated from the ring-breaking activity by heating the crude extract or by ammo- nium sulfate fractionation. The ring-breaking activity is lost by these treatments while the hydrolase activity remains intact. It has not been possible to obtain ring-breaking activity free of hydrolase activity.

Properties of Cyclopropane Ring-breaking System-The con- version of cyclopropanecarboxylic acid-X to y-hydroxybutyric acid is markedly dependent on pH. As shown in Fig. 6 the op- timal pH for the formation of y-hydroxybutyric acid is pH 6.7. It may be seen that the formation of y-hydroxybutyric acid and

0 10 20 30 40 50 60 0

Protein cmg)

FIG. 8. The dependence of cyclopropanecarboxylic acid-X metabolism on protein concentration. The enzyme solution used in this experiment was prepared in 0.1 M potassium phosphate buffer, pH 6.7, and contained 11.3 mg of protein per ml. Aliquots of this solution were diluted in 25-ml Erlenmeyer flasks to 6.0 ml with 0.1 M potassium phosphate buffer at pH 6.7. A 0.6.ml ali- quot of the radioactive cyclopropanecarboxylic acid-X solution described in the legend to Fig. 6 was added to each flask. In a con- trol flask the protein solution was replaced by an equivalent volume of buffer. All flasks were incubated for 13 min in a Dub- noff Metabolic Shaker at 30”. The incubations were terminated and samples prepared for and analyzed by Sephadex G-10 chroma- tography as described in the legend to Table I. After determining the total amount of radioactivity recovered from each column, the appropriate percentage of this total was assigned to cyclo- propanecarboxylic acid-X, cyclopropanecarboxylic acid, r-hydrox- vbutvric acid, and 1?1. The over-all recovery of radioactivitv from”the columns ranged from 72 to 77%. &HB, r-hydroxybu- tyric acid; CPCA, cyclopropanecarboxylic acid.

cyclopropanecarboxylic acid exhibits a reciprocal relationship which perhaps reflects the differences in the pH optima for the “hydrolase” activity and the ring-breaking activity. At this optimal pH the conversion of y-hydroxybutyric acid to end prod- ucts is also minimized. At the optimal pH the formation of y-hydroxybutyric acid is dependent on both the time of incuba- tion and protein concentration in the reaction mixtures. These relationships are shown in Figs. 7and 8, respectively.

The ring-breaking activity exhibits a partial requirement for low molecular weight heat-stable compounds. If the crude extract is subjected to gel liltration on Sephadex G-25, approxi- mately 60% of the catalytic activity is lost. The loss in cat- alytic activity may be restored by the addition of the superna- tant solution from the crude extract which had been boiled for 2 min. The lost activity could not be restored by a mixture of DPNH, TPNH, and ATP at final concentrations of 8 x 10m4, 7.6 X 1W4, and lOma M, respectively. The requirement was also not satisfied by a combination of TPNf, DPN+, FAD, FMN, ascorbate, metal ions, and tetrahydro-5,6-dimethylpteridine.

The ring-breaking activity is extremely labile. Incubation of the extract at 30” for 25 min in 0.1 M potassium phosphate buffer, pH 6.7, results in complete loss of activity. Dithiothreitol at a final concentration of 1W M did not prevent the loss of activity nor could it restore the lost activity. Although boiled extract could restore the activity lost by Sephadex G-25 chromatography it could not restore the activity of the inactivated enzyme system.

DISCUSSION

The fungus F. oxysporum Schlectendahl is capable of utilizing cyclopropanecarboxylic acid as its sole source of carbon and

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Issue of November 10, 1970 J. G. Xchiller and A. E. Chung 5863

energy. The organism must therefore degrade the cyclopropane ring to fulfill its metabolic requirements. By the use of pulse- labeling experiments with cyclopropanecarboxylic acid-1-14C it was shown that this compound was converted to a derivative of cyclopropanecarboxylic acid of unknown structure, y-hydroxy- butyric acid, and a number of other products. The data ob- tained from experiments with whole cells and cell-free extracts indicate that the sequence of reactions involved in the degrada- tion of cyclopropanecarboxylic acid is (a) an “activation” of cyclopropanecarboxylic acid to cyclopropanecarboxylic acid-X, (b) the cleavage of the cyclopropane ring in cyclopropanecar- boxylic-X by the formal addition of water across the ring and loss of the “activating” group with the resultant formation of y-hy- droxybutyric acid, and (c) the further metabolism of y-hydroxy- butyric to polar products.

Formation of cyclopropanecarboxylic acid-X has been observed only in intact cells. Sonic disruption of these cells results in a complete loss in the catalytic activity required for synthesis of this compound. Assuming an analogy between the synthesis of cyclopropanecarboxylic acid-X and the activating system for fatty acids prior to their degradation, cell-free extracts of Fusar- ium were incubated in the presence of cyclopropanecarboxylic acid-lJ4C, coenzyme A, ATP, and magnesium ions. No forma- tion of cyclopropanecarboxylic acid-X was observed. At- tempts have been made to identify the X moiety of cyclopropane- carboxylic acid-X.

The cyclopropanecarboxylic acid-X molecule has been obtained in radiopure form. This is shown in part by the fact that radio- active cyclopropanecarboxylic acid can be quantitatively recov- ered from solutions of cyclopropanecarboxylic acid-X that have been subjected to acid, basic, or enzymatic hydrolysis. Our data, at present indicate that the X moiety of cyclopropanecarbox- ylic-X is not coenzyme A. The absorbance at 280 rnp of a solu- tion of cyclopropanecarboxylic acid-X in which the concentration of bound cyclopropanecarboxylic acid was calculated from its specific activity was too low to accommodate a 1: 1 M ratio of cyclopropanecarboxylic acid to coenzyme A. The molecular weight of cyclopropanecarboxylic acid-X as determined by gel filtration and permeability through a diaflo membrane was less than 500. A coenzyme A moiety would yield a molecule larger than 500. The molecule is adsorbed on Dowex 50, a cation ex- change resin, but is not adsorbed by Dowex 1, an anionic resin, which indicates the possibility of a molecule with a positive charge; a coenzyme A derivative might be expected to be nega- tively charged.

Attempts to volatilize cyclopropanecarboxylic acid-X for mass spectrometry have been unsuccessful. Of further interest is the observation that cyclopropanecarboxylic acid-X does not give a ninhydrin-positive reaction which rules out a free amino group. The conversion of cyclopropanecarboxylic acid-X to y-hydroxy- butyric acid involves at least two distinct reactions (a) the formal addition of water across the ring to give a derivative of y-hydroxy- butyrate and (b) the removal of the X moiety. These processes may occur simultaneously or in a sequential manner; however, our present data do not yield any information on these points. The enzyme system described, however, offers an exciting oppor- tunity to study the mechanism of this novel ring cleavage reac- tion. Four hypothetical pathways for the over-all ring-open- ing reaction are shown in Fig. 9. Pathway a requires an or,/3 dehydrogenation similar to the type found in fatty acid oxidation (19). The product, L\I, fi-dehydrocyclopropanecarboxylic acid-X,

FIG. 9. Hypothetical pathways proposed to account for the degradation of cyclopropanecarboxylic acid-X by Fusarium cells.

then undergoes a Michael-type addition (20) of water across the (Y , P-unsaturated bond to yield P-hydroxycyclopropanecarbox- ylic acid-X. This step is analogous to the enoyl hydrase (19) re- action also encountered in fatty acid oxidation. The next step consists of the cleavage of a carbon-carbon single bond by the addition of 2 hydrogen atoms. No biochemical precedent is readily available for this type of reaction. The final step is the hydrolysis of y-hydroxybutyric acid-X to y-hydroxybutyric acid. Pathway b requires the participation of molecular oxygen in order to degrade the cyclopropane ring. This pathway re- quires an oxidation (hydroxylation) at a saturated carbon atom as its first step to yield the 3-hydroxy derivative of cyclopropane- carboxylic acid-X. This type of reaction is observed in the (Y hydroxylation of fatty acids (21), the w hydroxylation of fatty acids (19) and the steroid 6-@-hydroxylase reaction (19). The next step would yield y-hydroxybutyric acid-X which would then be hydrolyzed to free y-hydroxybutyric acid. The third Path- way c involves the simple addition of water in order to break a carbon-carbon single bond and yield y-hydroxybutyric acid-X. Although this would be a unique biochemical reaction it may be justified on the basis that cyclopropanecarboxylic acid esters be- have like ar,&unsaturated esters when attacked by nucleophiles (22) (i.e. they undergo a Michael addition). In this instance the nucleophile might be either the hydroxyl ion or water. The re- maining Pathway d requires that the cyclopropylcarbonyl moiety be joined to X in an oxygen ester linkage. This being the case, an internal rearrangement might occur that caused the oxygen atom in this linkage to become the ring oxygen of y-butyrolac- tone. Hydrolysis of the lactone would yield free y-hydroxy- butyric acid. Although Pathways a, b, and c require the exist- ence of y-hydroxybutyric acid-X, this intermediate has not been isolated. This may be rationalized by assuming that its rate of hydrolysis is extremely fast compared to its rate of formation. Similarly, y-butyrolactone has not been isolated. It too might suffer a relatively rapid hydrolysis.

A subsequent paper describes a series of experiments concerned with the mechanism by which this novel ring cleavage reaction proceeds.

The conversion of cyclopropanecarboxylic acid to y-hydroxy-

OH

A/ \ ?l,x “20, !,x A/

-2+ y p,,

A/ i9 H20

OH

‘X 6. * L i9 ‘X

+H2O d I -x ’ 1 +H20

-X

$0 \ G 0

H20 ) vAOH

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Metabolism of Cyclopropanecarboxylic Acid Vol. 245, No. 21

butyric acid yields an intermediate which can be readily utilized by a variety of possible metabolic pathways. Among the path- ways possible are (a) the oxidation of y-hydroxybutyric acid to succinic acid, (b) isomerization to /3-hydroxybutyrate, and subse- quent oxidation or transamination. It might be mentioned that the intact organism readily utilizes y-hydroxybutyric acid as its only source of carbon and energy.

Achwwledgmenk--We wish to thank Mr. J. Naworal for re- cording the rnms spectra and Dr. I. M. Campbell for helpful dis- cussions concerning the interpretation of the spectra. The mass spectrometric facilities are supported by Grant RR-00273 from the National Institutes of Health.

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Julian G. Schiller and Albert E. ChungThe Metabolism of Cyclopropanecarboxylic Acid

1970, 245:5857-5864.J. Biol. Chem. 

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