326 Biochimica et Biophysica Acta, 677 (1981) 326-329 Elsevier/North-Holland Biomedical Press

BBA 21570

BBA Report

STUDIES ON THE BIOSYNTHESIS OF N-(7-L-GLUTAMYL)-4-HYDROXYANILINE IN A GARICUS BISPOR US

IDENTIFICATION OF THE POSITION IN SHIKIMIC ACID AT WHICH THE AMINATION OCCURS

HIDEAKI TSUJI, NORIKO BANDO, TADASHI OGAWA and KEI SASAOKA

Department o f Nutrition, School of Medicine, Tokushima University, Kuramoto-cho, Tokushima 770 (Japan)

(Received May 18th, 1981)

Key words: Shikimic acid," Glutamyl-hydroxyaniline; 4-Hydroxyaniline; (Agaricus bisporus)

Labelled shikimic acid was efficiently incorporated into the aniline moiety of N-(7-L-glutamyl)-4-hydroxyaniline, a characteristic aromatic compound of the common mushroom,Agaricus bisporus. Incubations with [3-3H]-and [ 1,6-14C]shikimic acid clearly proved that the amination of shikimie acid occurs at its 4-position during the bio- synthesis of N-(7-L-glutamyl)-4-hydroxyaniline.

The common edible mushroom, Agaricus bisporus, contains novel aromatic compounds [ 1 ]. We have ear- lier followed the fate of labelled shikimic acid (see compound I, Fig. 1) in the fruiting bodies of the mushroom and we showed that it was actively me- tabolized to form several unidentified metabolites [1]. Among these metabolites, N-(7-L-glutamyl)-4- hydroxyanlline (compound II, Fig. 1), N-(7-L-gluta- myl)-3,4-dihydroxyaniline and L-3,4-dihydroxy- phenylalanine were identified and the radioactivity was showed to be located in the aniline moiety of the first two compounds [ 1,2]. In the present study, the position in shikirnic acid at which the amination occurs in the biosynthesis of 4-hydroxyaniline (com- pound III, Fig. 1) of N-(7-L-glutamyl)-4-hydroxy- aniline was examined using [3-all] - and [1,6-14C]- shikimic acid.

Freshly harvested fruiting bodies of the mushroom (in stage 2, see Ref. 3) were incubated with variously labelled shikimic acid as described previously [1]. (-)-[G-14C]Shikimic acid (81.1 Ci/mol) was pur- chased from New England Nuclear (Boston, MA, U.S.A.). By the method of Larsen [4], this com-

pound was confirmed to be uniformly labelled. (-)- [3-3H] Shikimic acid (56 Ci/mol) was prepared from the methyl ester of 3-dehydroshikimic acid [5,6] and NaBaH4 by the method of Leduc et al. [7]. NaBaH4 (10 Ci/mmol)and (-+)-[1,6-14C]shikimic acid (13.9 Ci/mol) were obtained from Commissariat a L'Ener- gie Atomique (Saclay, France). After incubation, labelled N-(7-L-glutamyl)-4-hydroxyanlline formed was isolated from the fruiting bodies as described ear- lier [1] and was recrystallized to constant specific activity from water. The pure, labelled specimens were diluted with unlabelled N-(7-L-glutamyl)-4- hydroxyanlline, which had been synthesized from 4-benzyloxyaniline and N-phthalyl-L-glutamic anhy- dride (unpublished data), and were hydrolyzed as described earlier [1]. 4-Hydroxyaniline, formed by hydrolysis, was used for the degradation experiments summarized in Fig. 1.

In the previous paper [ 1 ], active incorporation of the radioactive label from [G-14C]shikimic acid into the 4-hydroxyanlline moiety of N-(7-L-glutamyl)-4- hydroxyaniline was reported. When [3-3H]-and [G-x4c]shikimic acid were simultaneously incubated,

0304.4165/81[0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

327

i • 2H

He J : OH 614 I

1

.,H co CH=CHeCHCO2H n kH= 1

"5 3 5

2 6

NH z Ill

NHCOCH s NHCOCH a 0 IV V vI VII v m

I l l )

~COCH3 .~COCH3

(b).._(.(.(_~._~ ~ C e r 3NO= (J~ CH3NH2 (e~.~) ~NCH.I

NHCOCH 3 NHCOCH 3 u IX X Vl vB V=

~ . ~ O~ co.. co=. o~ (g) (h)

C(CH3) 3 ~(CH3) 3 CICH3) 3 X! Xlt XgI XlV XV

•.• ~ ~ ~ J;O2H

y v 3 " v co , .

XVI x v n x v m x l x

Carbon atom Degradation derived fromm procedure

C - 3 + C - 5 A

C-2 + C - 6 B

C-I and C-2 ÷ C-I and C-6

C- I * C - 4 D

Fig. 1. Degradation of 4-hydsoxyaniline. The products formed in the degzadation reactions were identified by their melting POints and spectroscopic behaviours. The reactions used: (a), acetylafion with acetic anhydride [ 8]; (b), nitration [ 9]; (c), hypobromite degxadation [ 10 ] ; (d), reduction with a Fe-HCI system [ 10 ]; (e), condensation with phthalic anhydride [ 11] ; (39, acetylation with acetic anhydride [ 12] ; (g), diazotization [ 13 ] ; (h), reduction with NaBI~ [ 13 ]; 03, alkTlation with isobutanol [ 14 ]; (/3, oxida- tion with KMnO4 [14]; (k), condensation with 2,4-dinitrophenylhydrazine [14]; (l), oxidation with V2Os [15]; (m), condensa- tion with l~-butadiene and oxidation with Na~ Cr2 07 [15]; (n), oxidation with KMnO4 [ 15 ]; (o), decaxboxylation with a Cu- quinoline system [ 15]. The compounds written in Roman num~als: I, shikirnic acid; II, N-(7-L-glutamyl)-4-hydroxyaniline; III, 4-hydroxyaniline; IV, N-acetyl-4-hydroxyaniline; V, 3,5-dinitzo-N-acetyl-4-hydroxyaniline; VI, bromopicrin; VH, methylamine; VIII, N-methylphthalimide; IX, N,O-diacetyl-4-hydsoxyaniline; X, 2,6-dim~o-N,O-diaceWI-4-hydroxyaniline; XI, 4-hydroxy- benzene diazonium borofluoride; XII, phenol; XIII, 4-tert-butylphenol; XIV, trimethylpyruvic acid; XV, 2,4-dinitrophenylhy- drazone of tximethylpyruvic acid; XVI, 1,4-benzoquinone; XVII, 1,4-naphthoquinone; XVIII, phthalic acid; XIX, barium car- bonate .

both the SH and 14C were efficiently incorporated into N-(7-L-glutamyl)-4-hydroxyaniline, and they were quantitatively located in the 4-hydroxyaniline moiety. The SH:14C ratio in shikimic acid was 1 : 0 . 8 2 , but the ratio changed to 1 : 0 . 7 4 in 4-hy- droxyaniline, indicating the loss of the carboxyl group of shikimic acid [4]. This result suggests that [3-SH]shikimic acid was incorporated into 4-hy- droxyaniline at the same rate with [G-14C]shikimic acid and ~H on C-3 o f shikirnic acid was retained in the shikimate-4.hydroxyaniline conversion.

To determine the location of SH in 4-hydroxy- aniline, its acetyl derivatives were nitrated, as shown

in degradation procedures A and B in Fig. I . In Table I, it can be seen that 3,5-dinitro-N-acetyl-4- hydroxyaniline (compound V) had the same specific activity as that of 4-hydroxyanfline, whereas 2,6- dinitro-N,O-diacetyl-4-hydroxyaniline (compound X) lost its radioactivity. These results show that SH was located at one of the positions ortho to the amino group of 4-hydroxyaniline (either C-2 or C-6). This means that the amination occurs at either C-2 or C-4 of shikimic acid during the shikimate-4-hydroxy- aniline conversion.

In the case o f 4-hydroxyaniline derived from [1,6- 14C]shikimic acid, there are three possibilities as to

328

TABLE I

LOCALIZATION OF THE TRITIUM ATOM IN 4-HYDROXYANILINE DERIVED FROM [3-3H]SHIKIMIC ACID

A fruiting body was incubated with 10/zCi of [ 3-3H] shikimic acid. Labelled N-(~-L-glutamyl)-4-hydroxyaniline was isolated from ten fruiting bodies. The radioactivity was measured with an Aloka LSC-700 liquid scintillation system as described previously [16]. The Roman numerals used in Fig. 1 are written in the parentheses. Relative activity = (the specific activity of the products/ the specific activity of 4-hydroxyaniline) × 100.

Compound Specific activity Relative activity (mCi/mol) (%)

N-(7-L-Glutamyl) -4-hydroxyaniline (II) 4-Hydroxyaniline (III) N-Acetyl-4-hydroxyaniline (IV) 3,5 -Dinitr o-N-acetyl-4-hydroxyaniline (V) N,O-Diacetyl-4-hydroxyaniline (IX) 2,6-Dinitro-N,O-diacetyl-4-hydroxyaniline (X)

31.09 30.10 100 29.79 99.0 29.83 99.1 29.09 96.6

0.40 1.3

the distribution of the labelled carbon atoms in 4-hy-

droxyaniline, because of the symmetrical structure of this compound: The first possibility is that either C-1

and C-2, or C-1 and C-6, are labelled. The second is that either C-2 and C-3, or C-5 and C-6, are labelled. The third possibility is that the radioactivity exists at

either C-3 and C-4, or C-4 and C-5.

In degradation procedure C (Fig. 1), the 2,4- dinitrophenylhydrazone of trimethylpyruvic acid

(compound XV), containing either C-1 and C-2 or C-1 and C-6 of 4-hydroxyaniline, was practically un- labelled (Table II). Therefore, the first possibility is

TABLE II

DEGRADATION EXPERIMENTS OF 4-HYDROXYANILINE DERIVED FROM [1,6 -14C] SHIKIMIC ACID

A fruiting body. was incubated with 10/zCi of [ 1,6 -14C]shikimic acid. Labelled N-(~/-L-glutamyl)-4-hydroxyaniline was isolated from ten fruiting bodies. The Roman numerals used in Fig. 1 are written in the parentheses. Relative activity = (specific activity of the products/the specific activity of 4-hydroxyaniline) × 100.

Compound Specific activity Relative activity (mCi/mol) (%)

N-(~,-L-Glu tamyl)-4-hydroxyaniline (II) 22.04 4-Hydroxyaniline (III) 21.20 2,4-Dinitrophenylhydrazone of trimethylpyruvic acid (XV) 0.42 3,5 -Dinitro-N-acetyl-4-hydxoxyanillne (V) 20.28 N-Methylphthalimide (VIII) b 4.96 2,6-Dinitro-N,O-diacetyl-4-hydroxyaniline (X) 20.82 N-Methylphthalimide (VIII) c 0.04 1,4-Naphthoquinone (XVII) 19.87 Phthalic acid (XVIII) 15.26 BaCO 3 (XIX) 4.93 d

100 2.0 a

9517 23.4 98.2 0.2

93.7 72.0 23.3

a Alkylation at either C-3 or C-5 of 4-hydxoxyaniline by isobutanol occurred in a slight degree, and a small amount of 2,4-dinitro- phenyihydrazone of trimethylpyruvie acid derived from trimethylpyruvic acid containing either C-3 or C-5 of 4-hydroxyaniline was formed [17].

b N-Methylphthalimide obtained from 3,5-dinitro-N-aeetyl-4-hydroxyaniline in degradation procedure A in Fig. 1. c N-Methylphthalimide obtained from 2,6-dinitro-N,O-diacetyl-4-hydroxyaniline in degradation procedure B in Fig. 1. d The radioactivity was measured in a solid state with the scintillation system [16], and the value was corrected by the use of

standard Ba 14CO 3.

eliminated. In degradation procedure A, when either C-3 or C-5 of 4-hydroxyanlline is labelled, 3,5-dinitro- N-acetyl-4-hydroxyaniline (compound V) should give equimolecular amounts of labelled and unlabelled bromopicrin (compound VI) by the hypobrornite degradation [10] and the specific activity of the resulting N-methylphthalimide (compound VIII) is expected to be one-quarter of that of 4-hydroxy- aniline. This expected value was obtained (Table II). On the other hand, in degradation procedure B,

N-methylphthalimide, obtained from 2,6-dinitro-N,O- diacetyl-4-hydroxyaniline (compound X), had no radioactivity, indicating that both C-2 and C-6 of 4-hydroxyaniline were unlabelled. These results rule out the second possibility and also support the result obtained in degradation procedure C. In degradation procedure D, the specific activity of phthalic acid (compound XVIII), obtained from 1,4-naphtho- quinone (compound XVII), was three-quarters of that of 4-hydroxyaniline. This indicates that one of the labelled carbon atoms of [1,6-14C]shikimic acid was located at C-2, C-3, C-5 or C-6 of 4-hydroxyaniline and was lost in the formation of phthalic acid. The specific activity (Table II) of barium carbonate (com- pound XIX), obtained from phthalic acid, shows that another labelled carbon atom was present at either C-1 or C-4 of 4-hydroxyanillne. It was proved by degradation procedures B and C that C-l, C-2 and C-6 were unlabelled. Therefore, the labelled carbon atoms should exist at either C-3 and C-4 or C-4 and C-5.

The results obtained by the above degradation experiments support the third possibility. In the experiments using [3-3H]shikimic acid, it was demon- strated that the amino group is attached to either C-2 or C-4 of shikimic acid. If C-2 of shikimic acid partici- pates in the amination, either C-2 or C-6 of 4-hy- droxyaniline should be labelled. The results obtained by degradation procedures B and C, however, exclude this possibility.

The results obtained in these experiments clearly demonstrate, for the ftrst time, that in the biochemi- cal shikimate-4-hydroxyaniline conversion in the mushroom, the amination occurs at C-4 of shikimic acid analogously to the biosyntheses of p-amino-

329

benzoic acid [18] and p-aminophenylalanine [19,20]. Therefore, the present work is important in under- standing the mechanism of the biosynthesis of 4-hy- droxyaniline.

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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