THE ENZYMATIC CONVERSION OF ANTHRANILIC ACID TO INDOLE*

BY CHARLES YANOFSKY

(From the Department of Microbiology, School of Medicine, Western Reserve University, Cleveland, Ohio)

(Received for publication, March 26, 1956)

Studies with various microorganisms have implicated anthranilic acid and indole as intermediates in the biosynthesis of tryptophan (l-5). The mechanism of tryptophan synthesis from indole has been established (6-g), but relatively little is known about the conversion of anthranilic acid to indole. Isotope studies on the synthesis of indole from anthranilic acid have shown that the carboxyl carbon of anthranilic acid is lost during this conversion (10, 11) and that the 2 carbon atoms which complete the pyr- role ring of indole are probably derived from C-l and C-2 of a ribose de- rivative (11). Enzymatic investigations have implicated 5-phosphori- bosyl-1-pyrophosphate’ as the actual source of the 2 carbon atoms and have led to the isolation of an intermediate, indole-&glycerol phosphate, in the conversion of anthranilic acid to indole (12). The present report is con- cerned with the enzymatic conversions of anthranilic acid to indole-3-glyc- erol phosphate and of indole-&glycerol phosphate to indole.

Methods

A tryptophan auxotroph of the K-12 strain of Escherichia coli, strain T-3, was employed as the source of the enzymes catalyzing the conversion of anthranilic acid to indole. This mutant is blocked in the synthesis of anthranilic acid, and thus its tryptophan requirement can be satisfied by anthranilic acid or indole. The conditions of growth used and the method of preparation of crude extracts of this mutant were essentially the same as described previously (11). The mutant was grown on a minimal medium (13), supplemented with anthranilic acid (2 y per ml.) and 0.16 per cent glucose. Cultures were incubated with shaking for 40 hours at 30”. The cells were harvested by centrifugation, washed once with saline, and sus- pended in 0.1 M phosphate buffer at pH 7.0. These washed suspensions were then disrupted in a 9 kc. Raytheon sonic oscillator and centrifuged

* This investigation was supported by the National Science Foundation and was performed during the tenure of a Lederle Medical Faculty Award to the author.

1 The following abbreviations are employed in this paper: IGP, indole-3-glycerol phosphate; IG, indole-3-glycerol; PRPP, 5-phosphoribosyl-1-pyrophosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; R5P, ribose-5-phosphate; Pi, inorganic phosphate; Tris, tris(hydroxymethyl)aminomethane.

171

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172 ANTHRANILIC ACID CONVERSION TO INDOLE

twice to remove debris for 20 minute periods at 60,000 X g in a Spinco pre- parative centrifuge. The supernatant solutions obtained contained ap- proximately 35 mg. of protein per ml. These crude extracts, when suitably supplemented, catalyzed the complete conversion of anthranilic acid to indole (14). Ammonium sulfate fractionation of such preparations yielded two separate fractions, arbitrarily designated Fractions A and B, which catalyzed successive reactions in the transformation of anthranilic acid to indole.

Fraction A was prepared as follows: 18.7 gm. of solid ammonium sulfate were added for each 100 ml. of crude centrifuged extract of strain T-3. The mixture was allowed to stand in an ice bath for 20 minutes and then was centrifuged in the cold. The supernatant solution was ‘saved for the isolation of Fraction B, while the precipitate was used in the further prep- aration of Fraction A. The precipitate was dissolved in 0.1 M phosphate at pH 7.0 (30 ml. for every 100 ml. of extract) and dialyzed, with internal stirring, for 3 hours against 0.02 M phosphate at pH 7.8. The dialyzed preparation was treated with ammonium sulfate (4.3 gm. for each 40 ml.) and, after 20 minutes, was centrifuged in the cold. The precipitate was discarded and 3 gm. of ammonium sulfate were added to the supernatant solution. After 20 minutes, the precipitate was collected by centrifugation and dissolved in 15 ml. of 0.1 M phosphate buffer at pH 7.8. It was then dialyzed for 3 hours against 0.02 M phosphate buffer at pH 7.8 and stored at -15”. This preparation, designated Fraction A, catalyzes the conver- sion of anthranilic acid to IGP and is unable to convert the latter compound to indole.

The supernatant solution from the first ammonium sulfate precipitation was treated with an additional 9.5 gm. of ammonium sulfate and the pre- cipitate collected by centrifugation. This precipitate was dissolved in 30 ml. of 0.1 M Tris buffer at pH 7.8 and dialyzed against the same buffer (0.02 M) for 3 hours. The preparation obtained was then placed in a 50” water bath and stirred continuously for 20 minutes. After this treatment, the mixture was chilled rapidly and the precipitated protein removed by centrifugation. The supernatant solution was treated with ammonium sul- fate (8.6 gm. for every 30 ml.) and, after standing for 20 minutes, was cen- trifuged. The precipitate obtained was dissolved in 0.1 M Tris buffer at pH 7.8 and dialyzed for 3 hours against the same buffer (0.02 M). The final preparation, designated Fraction B, was stored at -15’. This fraction converts IGP to indole but does not catalyze the utilization of anthranilic acid.

Assay Methods

Disappearance of anthranilic acid was followed fluorometrically (15). Aliquots (0.05 or 0.1 ml.) were added to 1.0 ml. of 0.1 M phosphate buffer

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C. YANOFSKY 173

at pH 6.0 and read in a Farrand fluorometer. The assay range employed was 0.2 to 5 y of anthranilic acid.

Indole was determined calorimetrically with Ehrlich’s reagent (16) and pentose by the procedure of Umbreit et al. (17). Inorganic phosphate was determined by the method of Fiske and Subbarow (18).

IGP was assayed in several ways: qualitatively with a modified ferric chloride Salkowski reagent (19) or by enzymatic conversion to indole, or quantitatively by extinction measurements at 280 rnp or by oxidation with metaperiodate to indole-3-aldehyde. These methods are described below.

The ferric chloride assay was performed as follows. To 1 ml. containing IGP or IG, 1.5 ml. of ferric chloride reagent (1 ml. of 0.5 M FeC13 plus 50 ml. of water plus 30 ml. of concentrated H2S04) were added. A pink to red color developed in about 3 minutes. As little as 10 y of IGP could be detected by this method. The intensity of t,he color produced was pro- portional to IGP and IG concentration, but the color did not persist in the presence of high salt concentrations. Nevertheless, this method had the advantage of being rapid and thus was employed in the isolation of IGP.

IGP was assayed eneymatically as follows: 0.1 to 0.3 ml. (5 to 50 r) of a solution of IGP was added to 0.2 ml. of 0.1 M phosphate at pH 6.0. An excess of Fraction B (0.05 ml.) was then added and the mixture incubated at 37” for 15 minutes. The reaction was terminated by addition of 0.1 ml. of 1 N NaOH and the indole formed was extracted with 2 ml. of toluene. An indole assay was performed by using Ehrlich’s reagent on a 1 ml. ali- quot of the toluene layer. Between 80 and 95 per cent of IGP was con- verted to indole by this method.

Neutral or alkaline aqueous solutions of IGP, when free from other ab- sorbing materials, were standardized in a Beckman spectrophotometer at 280 mp. The 280:260 and 280:240 ratios (1.32 and 3.1, respectively, for IGP) were used to assess the purity of IGP during isolation.

The preferred method of IGP assay was based on oxidation to indole- 3-aldehyde as follows: 0.1 ml. of 1 M acetate buffer at pH 5.0 and 0.5 ml. of 0.1 N sodium metaperiodate were added to 0.4 ml. of a solution of IGP and the mixture was incubated at room temperature for 20 minutes. 0.25 ml. of 1 N NaOH was then added and the indole3-aldehyde formed was ex- tracted with 5 ml. of ethyl acetate. The ethyl acetate layer was clarified by a 1 minute centrifugation, and its indole aldehyde content was deter- mined in a Beckman spectrophotometer at 290 mp. An IGP sample, previously standardized by absorption at 280 mp, and an indole-3-aldehyde sample were similarly treated and employed as standards. If the IGP sam- ple contained indole, the indole was first extract,ed with 3 ml. of toluene. Aliquots of the aqueous layer were then removed for IGP assay.

The aldolase employed was purchased from the Nutritional Biochemicals Corporation.

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174 ANTHRANILIC ACID CONVERSION TO INDOLE

Results

Conversion of Anthranilic Acid to Indole Glycerol Phosphate-Following separation of the enzymes involved in the conversion of anthranilic acid to indole into two fractions, the characteristics and requirements of the frac- tions were investigated. Fraction A catalyzes the following interconver- sion :

Anthranilic acid + PRPP + + + indole-a-glycerol phosphate

The requirements for this sequence of reactions are shown in Table I. It can be seen in Experiment A, Table I, that PRPP and Mg++ ions are re-

TABLE I Requirements for Anthranilic Acid Utilization

Experiment A I Experiment B

Disa pearance of ant R ranilic acid

Supplements

10 min. 20 min. ~-

plole imkdc

Complete system 0.12 0.2 Minus PRPP 0 0

“ %++ 0.02 0.03

Complete system Minus ATP

L‘ R5P

“ Mg++

Disappearance of anthranilic acid

10 min. 20 min. _-

~molc ~mole

0.08 0.14 0 0 0 0 0 0.01

In Experiment A, each tube contained 0.22 pmole of anthranilic acid, 0.1 ml. of 0.5 M phosphate buffer at pH 7.8, and 0.03 ml. of Fraction A in a final volume of 0.6 ml. PRPP (0.24 pmole) and Mg++ (1 Nmole) were added, except as indicated.

In Experiment B, each tube contained anthranilic acid, buffer, and enzyme as above, and, in addition, ATP (1.2 amoles), R5P (0.5 pmole), and Mg++ (1 Nmole), except as indicated.

quired for anthranilic acid utilization. Little or no reaction occurs when either of these supplements is omitted. The requirement for PRPP can be satisfied by supplying both ATP and R5P (Experiment B, Table I) ; however, with the latter supplements, anthranilic acid disappears at a some- what lower rate, as is evident from the rate data in Fig. 1. Disappearance of anthranilic acid is related to the amount of pentose supplied, whether this pentose is in the form of R5P or PRPP. In two experiments in which all components except R5P were present in excess, 0.26 and 0.22 pmoles of anthranilic acid disappeared when 0.25 and 0.21 pmoles of R5P were sup- plied. In a similar experiment in which 0.24 pmole of PRPP was limiting, 0.2 pmole of anthranilic acid disappeared. Furthermore, in the presence of Fraction A, both pentose (as PRPP) and anthranilic acid disappear at ap- proximately the same rate. This can be seen in Fig. 2. It can also be

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C. YANOFSKY 175

80

60

L 0 IO 20 30

MINUTES

FIG. 1. Comparison of the rate of anthranilic acid utilization in the presence of PRPP or ATP plus R5P. Both tubes contained 0.44 pmole of anthranilic acid, 100 ltmoles of phosphate at pH 7.8,2 pmoles of MgSO,, and 0.06 ml. of Fraction A. One tube contained 0.96 pmole of PRPP while the second contained 1.2 rmoles of ATP and03pmole of R5P. The final volume was 1.2 ml.

PM 1 0 ANTHRANILIC ACID 2,0 _ l PENTOSE .

- 0.4

-0 I I 0 IO 20 30 45 60

MHUTES

FIQ. 2. Comparison of anthranilic acid and pentose disappearance and indole formation. The reaction mixture contained 2.2 amoles of anthranilic acid, 3.6 pmoles of PRPP, 1 mmole of phosphate buffer at pH 7.8, 10 pmoles of MgSO,, and 0.3 ml. of Fraction A in a final volume of 6 ml. Aliquots were removed at the times indicated and assayed for anthranilic acid, pentose (0.5 ml. aliquots were treated with 1 ml. of 10 per cent perchloric acid, the precipitate was removed, and portions of the supernatant solution were assayed), or indole.

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176 ANTHRANILIC ACID CONVERSION TO INDOLE

seen that indole is not formed in the presence of Fraction A but is formed when Fraction B is added to the reaction mixture.

In several experiments, disappearance of anthranilic acid was compared with IGP formation; the results of two typical experiments were as follows: 0.16 and 0.18 pmoles of anthranilic acid disappeared, while 0.13 and 0.16 pmoles of IGP were formed.

In addition to the requirements for anthranilic acid utilization, men- tioned above, there is an absolute requirement for Pi when ATP and R5P are substituted for PRPP. With PRPP there is no requirement for Pi; however, if either ATP or ADP is added to a reaction mixture containing PRPP, anthranilic acid uptake is inhibited. This inhibition is relieved by inorganic phosphate. The experiments described below and performed with P32-labeled inorganic phosphate have excluded Pi as a precursor of the phosphate group of IGP. Our present interpretation of these findings is that Pi relieves the ATP or ADP inhibition of the reaction between anthranilic acid and PRPP.

Origin of Phosphate Group of Indole Glycerol Phosphate--Isotope experi- ments were performed with P32-labeled Pi and PRPP-5-P32 to determine the origin of the phosphate group of IGP. Fraction A was incubated with labeled PRPP and unlabeled Pi in the presence of anthranilic acid until most of the anthranilic acid disappeared. The reaction mixture was then heated to precipitate protein, was centrifuged, and the supernatant solu- tion chromatographed on paper, a developing solvent containing methyl alcohol, ethyl acetate, and water (1:2: 1 by volume) being used. Identical samples incubated with labeled Pi and unlabeled PRPP were also chro- matographed in this manner. Radioautographs of the chromatograms were prepared and the position and shape of the radioactive spots were compared with IGP spots (developed by spraying the paper with ferric chloride reagent). The results of these experiments clearly showed that the phosphate group of IGP was derived from the terminal phosphate of PRPP and not from Pi.

Xpecijicity of Fractions A and B-A number of substituted anthranilic acids, 3-methylanthranilic acid, 4-methylanthranilic acid, 5-methylanthra- nilic acid, and 5-fluoroanthranilic acid, were tested as possible substrates for the reaction catalyzed by Fraction A. Incubation mixtures compar- able to those in Table I were employed, with the substitution of an equi- molar amount of one of the above compounds for anthranilic acid. All of the substituted anthranilic acids except 3-methylanthranilic acid were at- tacked by Fraction A. This is shown in Fig. 3, from which it can also be seen that the reaction proceeded somewhat faster with 5-methyl- or 5&i- oroanthranilic acid as substrate than with anthranilic acid. Following in- cubation, ferric chloride reagent was added to the reaction mixtures con-

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C. YANOFSKY 177

taining the substituted anthranilic acids. All except the one containing 3-methylanthranilic acid gave the color reaction characteristic of IGP. To a similar set of tubes, Fraction B was added and the mixtures were reincu- bated. The contents of each were then extracted with toluene and aliquots of the toluene layer were assayed for indole-like compounds with Ehrlich’s reagent. Positive reactions were given by all except the reaction mixture containing 3-methylanthranilic acid. The indole-like compounds formed were presumably the substituted indoles corresponding to the substituted anthranilic acids employed. It was previously shown that, with cell sus- pensions of strain T-3, 4-methylanthranilic acid is converted to the cor- responding substituted indole, 6-methylindole (20).

20

W .I6

z E 9 .I2 Ek 25 5 06

E

‘i .04

I I 0 5 IO 15 20 25

MINUTES FIG. 3. Comparison of the rate of utilization of various substituted anthranilic

acids. 0, 5-fluoroanthranilic acid; 0, 5-methylanthranilic acid; A, anthranilic acid; A, 4-methylanthranilic acid; Cl, 3-methylanthranilic acid.

Isolation of Indole-S-Glycerol Phosphate-The following incubation mix- ture was employed for the formation of IGP: 0.75 mmole of anthranilic acid, 2 mmoles of ATP, 0.8 mmole of R5P, 1.7 mmoles of MgS04, 17 mmoles of phosphate buffer at pH 8.2, and 34 ml. of Fraction A in a final volume of 1080 ml. The mixture was incubated at 37” for 30 to 40 minutes. Ali- quots were removed every 5 to 10 minutes to follow the disappearance of anthranilic acid. At the end of the incubation period the mixture was chilled rapidly and 1 N acetic acid was added until the pH was lowered to 6.5 to 7.0. Several portions of Darco G-60 (acid- and alkali-washed) were then added to adsorb the IGP. After each addition, the Darco was re- moved by filtration and a portion of the filtrate assayed for IGP with ferric chloride reagent. The Darco treatment was discontinued when a nega- tive test for IGP was obtained. The charcoal was then washed once with water and the IGP eluted by stirring with 40 per cent alcohol containing

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178 -1sTIIRANILIC ACID CONVERSION TO INDOLE

5 ml. of concentrated NHdOH per liter. The eluate was freed from char- coal by filtration and was then concentrated in vacua to a small volume (20 to 30 ml.). This solution was adjusted to pH 8 to 8.5 with saturated Ba(OH)2 and a 25 per cent solution of barium acetate was added until further additions no longer produced a precipitate. The precipitate was removed by centrifugation and, before being discarded, was washed three times with small volumes (3.5 ml.) of water containing a few drops of Ba(OH)2. The washings were combined with the original supernatant solution and 1 ml. of barium acetate solution was added, followed by 2 volumes of acetone. The precipitate was collected by centrifugation and washed twice with acetone. The final precipitate contained most of the IGP. This precipitate was dissolved in water and applied to a 2 X 32 cm. Dowex 1 chloride (2 per cent cross-linked) column. The column was prepared by treating Dowex 1 chloride in the column successively with 200 ml. of 1 M NaCl (this and all subsequent solutions added to the column contained 0.1 ml. of 1 M NaOH per 100 ml.) and with 100 ml. of 0.01 M

NaOH. The IGP solution was then applied and the column again treated with 100 ml. of dilute alkali. It was essential that all solutions applied to the column were alkaline; otherwise the IGP was destroyed during isola- tion. The column was then washed with 600 ml. of 0.1 M NaCl. The IGP was eluted from the Dowex by gradient elution with 400 ml. of 0.1 M NaCl in the mixing flask and 0.5 M NaCl in the reservoir flask. 20 ml. fractions were collected and a sample from each was tested for IGP with ferric chloride reagent. The IGP was usually present in fractions 18 to 30. The purity of these fractions was determined by measuring their absorption at 240, 260, and 280 mp. Of the ten to twelve fractions con- taining IGP, the first three to five usually contained appreciable amounts of adenylic acid, while the last two fractions occasionally contained small amounts of ADP. The IGP fractions containing impurities were combined and the Darco and Dowex steps repeated. IGP fractions which were free of absorbing impurities were adjusted to pH 6.5 to 7 and passed through a 2 cm. X 5 cm. (diameter) column of Darco G-60. The IGP was adsorbed completely. The column was washed with water and finally the IGP was eluted with 40 per cent alcohol containing 1 ml. of concentrat,ed NH,OH per 100 ml. The eluate was concentrated in vucuo to about 5 ml. and 1 N acetic acid was added until the pH was lowered to about 6. This treat- ment precipitated a small amount of charcoal which always contaminated the eluate. The precipitate was removed by centrifugation and the super- natant solution adjusted to pH 8 to 8.5 with saturated Ba(OH)2. 1 ml. of a 25 per cent solution of barium acetate was added, and the barium salt of IGP precipitated by the addition of 4 volumes of alcohol and 2 volumes of acetone. The precipitated barium salt was washed twice with acetone

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C. YANOFSKY 179

and dried with air on a Biichner funnel. Elemental analyses and evidence for the identity of the isolated product have been presented elsewhere (12). The absorption spectrum of the barium salt of IGP is given in Fig. 4. It is similar to, but not identical with, that of indole.

Conversion of Indole Glycerol Phosphate to Idle-It has been already noted (see Fig. 2) that addition of Fraction B to a mixture previously in- cubated with Fraction A results in the production of indole. Incubation of isolated IGP with Fraction B also results in the production of indole,

FIG. 4. (200 Y per

I I I I t

230 250 270 290 310 w

Absorption spectrum of the barium salt of indole- 3 ml. in water).

-glycerol phosphate

with a yield of 80 to 95 per cent of theoretical. In one large scale experi- ment, the indole formed from IGP was isolated as the picrate to establish its identity unequivocally. The isolated picrate had the same melting point as an authentic sample of indole picrate (174-175”), and the mixed melting point was also the same.

Enzymatic hydrolysis of IGP would be expected to yield equimolar amounts of indole and 3-phosphoglyceraldehyde. Tests performed to detect triose phosphate during the conversion of IGP to indole indicated the presence of a compound which gives a 2,4-dinitrophenyl osazone (12) with an absorption spectrum identical with that of the 2,4-dinitrophenyl osazone formed by the triose phosphates (21). Alkali-labile phosphate also appears, thus further suggesting that triose phosphate is a reaction

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180 ANTHRANILIC ACID CONVERSION TO INDOLE

product. The results of two experiments in which indole formation and alkali-labile phosphate formation were compared are presented in Table II. It can be seen that in the absence of KCN approximately 40 per cent of the phosphate expected, on the basis of the indole formed, appears as alkali-labile phosphate, while in the presence of KCN there is fair agree- ment between indole formation and alkali-labile phosphate formation. Additional tests with Fraction B showed that this fraction contains a CN- sensitive phosphatase which is active on triose phosphate, generated from hexose diphosphate with aldolase.

TABLE II

Indole and Alkali-Labile Phosphate Formation from Indole Glycerol Phosphate

Without KCN With KCN

Experiment No.

Pi *;;fg Alkali- Indole Pi

*g+lf Alkali- Indok labile P formed labile P formed

___- ~___ ~mole pmolc pmle pmolc pnole jmlolcs /mole pnole

1 0.48 0.8 0.32 0.78 0.14 0.9 0.76 1.0 2 0.55 0.82 0.27 0.81 0.14 1.1 0.96 0.99

Each tube contained 1.5 pmoles of IGP, 0.25 ml. of Fraction B, and 0.1 ml. of 0.5 M Tris buffer at pH 7.8 in a final volume of 1 ml. As indicated, 0.2 ml. of a 0.2 M neutralized solution of KCN was also present. Incubation was at 37” for 25 minutes. 0.1 ml. of the incubation mixture was removed for indole assay and 0.4 ml. of 10 per cent trichloroacetic acid was added to the remainder. After removal of the precipi- tate, 0.5 ml. aliquots of the supernatant solution were analyzed for Pi and for alkali- labile phosphate (the aliquot was mixed with an equal volume of 2 N KOH and neu- tralized after 20 minutes).

Indole-S-glycerol, prepared by treating IGP with intestinal phosphatase, is not converted to indole by Fraction B. Maximal activity of Fraction B is obtained at about pH 6. The name “indole glycerol phosphate hydro- lase” is proposed for the enzyme which converts IGP to indole.

Indole Glycerol Phosphate Formation from Indole and Triose Phosphate- In view of the fact that the enzymatic hydrolysis of IGP rarely went to completion, an attempt was made to determine whether the reaction was reversible. Fraction B was incubated with indole, aldolase, and hexose diphosphate, and the reaction mixture was analyzed for indole disappear- ance and IGP formation. The results of a typical experiment are shown in Table III. It can be seen that Fraction B catalyzed the utilization of indole and the formation of IGP. To determine whether the reaction product was indeed IGP, an aliquot from a similar experiment was chroma- tographed on paper with the developing solvent employed previously, sup- plemented with 1 ml. of 1 N NH,OH per 166 ml. A spot reacting with

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C. YANOFSKY 181

ferric chloride appeared at the same Rp as IGP when the sample was chro- matographed alone or with added IGP. In addition, when the material present in a duplicate unsprayed spot was eluted and treated with meta- periodate, indole-3-aldehyde, identified by its characteristic absorption spectrum, was formed. It appears, therefore, that Fraction B catalyzes both the conversion of IGP to indole and 3-phosphoglyceraldehyde and the reverse reaction, the formation of IGP from indole and triose phosphate. Whether these two activities are due to the same enzyme or to two separate enzymes remains to be determined.

TABLE III Indole Glycerol Phosphate Formation j’ram Indole and Triose Phosphate

Indole disappearing

Complete system Minus Fraction B

“ aldolase “ hexose diphosphate “

indole

Jmolc

0.25 0 0 0

IGP formed

pmolc

0.19 0

0

Each tube contained 0.6 amole of indole, 15 moles of hexose diphosphate, 0.2 ml. of aldolase, 0.24 ml. of Fraction B, and 0.2 ml. of 0.5 M phosphate buffer at pH 7.8 in a final volume of 1.4 ml. Incubation was at 37” for 30 minutes. The residual indole was extracted with toluene, and indole assays were performed on aliquots of the toluene layer. An aliquot from the aqueous layer was assayed for IGP by meta- periodate oxidation.

DISCUSSION

The data presented in this paper indicate the following mechanism of indole biosynthesis in E. coli:

Anthranilic acid + PRPP + indole glycerol phosphate --f indole + triose phosphate

This scheme is also supported by the results of enzymatic and metabolite accumulation investigations.* These studies have shown that tryptophan auxotrophs of E. co& blocked in the conversion of anthranilic acid to indole, fall into two groups, according to the content of enzymes involved in the above reactions. One group specifically lacks one or more of the enzymes involved in IGP formation from anthranilic acid, while the second group contains these enzymes but lacks IGP hydrolase. The inability of these mutants to synthesize tryptophan associated with the absence of specific enzymes would appear to be strong evidence for the view that the reactions being considered represent the principal pathway of tryptophan synthesis

* C. Yanofsky, unpublished data.

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182 ANTHRANILIC ACID CONVERSION TO INDOLE

in E. co&i. Data on metabolite accumulation also lend support to this view. Mutants of the group lacking IGP hydrolase, those which might be expected to accumulate IGP, accumulate a compound which appears to be IG (the accumulated compound has the same RF in several solvent systems as IG formed from IGP by treatment with intestinal phosphatase). An unidentified compound with properties similar to IG has previously been reported to be accumulated by certain tryptophan auxotrophs of E. coli (22) and of Salmonella typhimurium (23). In addition, the group of mu-

I01 + HO -P-0-P-0-C-CHOH-CHOH-C-CH,0P03H2

I I H H OH OH

COOH / a I

HO-C-CHOH-CHOH-CH,0P03H2 COOH

II I01 \

N - C - CHOH - CHOH - C- CH,OPO,H, H H H

H

CHOH-CHOH-CH,0P03H,

H FIG. 5. Hypothetical scheme of IGP formation from anthranilic acid

tants unable to form IGP from anthranilic acid accumulate anthranilic acid. Thus accumulation by both groups of mutants is consistent with the postulated scheme of indole biosynthesis.

In growth tests performed with IGP and IG, neither compound was found capable of supporting the growth of auxotrophs which respond to anthranilic acid. This observation could be explained by assuming that E. coli is impermeable to IGP and that IG, on the other hand, cannot be rephosphorylated. In this connection, it may be noted that intermediates in histidine synthesis, imidazole glycerol and imidazole glycerol phosphate, are also incapable of supporting the growth of histidine mutants which might be expected to respond to one or both (24).

The data presented in this paper provide no clues as to the detailed

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C. YANOFSKY 183

mechanism by which anthranilic acid is converted to IGP. By analogy with other reactions in which PRPP participates (25, 26), it seems likely that the first step would involve the formation of the ribotide of anthranilic acid (see Fig. 5, for hypothetical scheme of IGP formation). This com- pound could be converted by a reaction similar to the Amadori rearrange- ment (27) to a 1-deoxy, 2-keto intermediate which, in the enol form, would have both the double bond of the pyrrole ring of indole and a hydroxyl group on the 2nd carbon atom of the side chain. The hydroxyl group would then be in position for ring closure with the carbon atom of the ben- zene ring to which the carboxyl group is attached. Finally, either ring closure followed by decarboxylation or decarboxylation followed by ring closure would give IGP.

In view of the present evidence for the participation of IGP as an inter- mediate in tryptophan synthesis, it is perhaps surprising that in the syn- thesis of this amino acid the 3-carbon side chain of IGP is removed and replaced from serine. IGP could conceivably give rise to tryptophan more directly by the sequence of reactions which appear to be involved in histi- dine synthesis (24) ; namely, the conversion of a glycerol phosphate side chain to an alanine side chain. In view of the data presented in this paper which suggest that the conversion of IGP to indole is an essential step in tryptophan synthesis, it seems unlikely that this pathway is operative in E. coli. It remains to be determined whether it is employed by other microorganisms.

SUMMARY

The mechanism of the enzymatic conversion of anthranilic acid to indole in Escherichia coli has been investigated. Ammonium sulfate fractionation of extracts of a tryptophan auxotroph of E. coli has provided two separate fractions which catalyze successive reactions in the conversion of anthra- nilic acid to indole. One fraction catalyzes the formation of indole-3- glycerol phosphate from anthranilic acid and 5-phosphoribosyl-l-pyrophos- phate. A procedure for the isolation of IGP has been developed. The second fraction converts indole glycerol phosphate to indole and triose phosphate and also catalyzes the reverse reaction, the formation of indole glycerol phosphate from triose phosphate and indole. The significance of these reactions in the biosyntheses of indole and tryptophan is discussed.

The author is indebted to Dr. D. Goldthwait for supplying the labeled and unlabeled samples of 5-phosphoribosyl-1-pyrophosphate and for many interesting and helpful discussions during the course of this work. It is also a pleasure to acknowledge the valuable technical assistance of Mrs. N. Deyczakiwsky.

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184 ANTHRANILIC ACID CONVERSION TO INDOLE

The substituted anthranilic acids were kindly supplied by Dr. F. Pilgrim of Chas. Pfizer and Company.

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Charles YanofskyANTHRANILIC ACID TO INDOLE

THE ENZYMATIC CONVERSION OF

1956, 223:171-184.J. Biol. Chem. 

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