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To cite this article: Tsutomu Hoshino, Tadao Kondo, Takeo Uchiyama & Nagahiro Ogasawara
(1987) Biosynthesis of Violacein: a Novel Rearrangement in Tryptophan Metabolism with
a 1,2-Shift of the Indole Ring, Agricultural and Biological Chemistry, 51:3, 965-268, DOI:
10.1080/00021369.1987.10868084
TABLE I. 'H AND 13C NMR DATA OF VIOLACEIN AND DEOXYVIOLACEIN IN DMSO-d6
Violacein Deoxyviolacein
Position
13C 'H 13C
'H and 13C NMR spectra were measured at 200 and 50.1 MHz, respectively. The chemical shifts of the 'H and 13C
NMR spectra, in [) (ppm), are from internal TMS. The parentheses refer to the multiplicity of signals and coupling
constants (Hz).
* J H , -H 2 . ** Long-range coupling constants 4 J H13-HlO' l3C-enriched deoxyviolacein: 1 J C ,2 -C13 = 54.8 H'z,
2 J C ll -C '4 =7.1 Hz.
and deoxyviolacein are shown in Table I, and tography, using Sephadex LH 20 with an
details of these assignments will be reported eluent of methanol; deoxyviolacein was fol-
elsewhere. lowed by violacein. Each blue fraction was
Violacein and deoxyviolacein were pro- concentrated to a small volume and was
duced during the cultivation of Chromobacte- allowed to stand overnight, whereupon blue
rium violaceum JCM 1249 in a nutrient broth crystalline pigments gradually separated out.
medium (at pH 7) consisting of meat extract The addition of 25 mg of tryptophan to
(3 g), polypeptone (5 g), sodium chloride (5 g) 1000 ml of medium increased the pigment pro-
and water (1000 ml), with agitation for 48 hrs duction by about l.5 fold.
on a rotary shaker at 180 rpm and 25C. Blue L-[3-13C]tryptophan (13C 94%, 5 mgf) was
bacterial cells were harvested by centrifugation administered to a 300 ml erlenmeyer flask con-
and the blue pigments were extracted several taining 100 ml of medium, which was then
times with methanol. The extract was ad- cultured under the conditions previously men-
sorbed on Amberlite XAD 2, which was then tioned (yield, 20mg of viol ace in and 3.5mg of
washed with hot water and 40% aq. methanol, deoxyviolacein from six flasks). High level
and subsequently eluted with hot abs. meth- enrichment of the i3C atoms was observed at
anol. Separ~tion of violacein and deoxy- both C12 and C13 in the proton-noise de-
violacein was achieved by column chroma- coupled 13C NMR of violacein (Fig. 2). Satel-
Biosynthesis of Violacein 967
12 13
1
J c_c (12-13)
11
1"
(B) [2_ 13Cl Trp fed
MJN;l<\!.f;I)..I$\.~"~~J."fii\\;li~~",:,\\!\f,i~~~v,w.M1W~4iI4,'>ll\"\lj\:~\\'iiV~~,'\\lJ.'i~~~
(C) Natural
FIG. 2. Proton-noise Decoupled 13C NMR Spectra of Labeled and Natural Violaceins (22.5 MHz).
Spectra were measured under identical instrumental conditions except for the number of accumulations.
lite peaks due to 13C_13C coupling of two positions of both Cll and Cl4 as shown in
contiguous 13C atoms were also observed as a Fig. 2 (note the enriched signals and the
result of the high incorporation rate of labeled satellite peaks arising from two-bond 13C_13C
tryptophan. These findings suggest that the coupling via a nitrogen atom). The 13C atom
carbon skeleton of the pyrrolidone ring was percentages at the labeled positions were close
formed by the condensation of the side chains to those of violacein produced by feeding with
of two tryptophan molecules. Anal;:ses of El- [3-13C]tryptophan.
Ms and the ratio between the satellite peaks In the case of deoxyviolacein, identical in-
and the central peak inthe l3C NMR spectrum corporation patterns and almost the same
are indicative of the following isotopic dis- incorporation percentage of the isotope as
tribution: 12C 12-12 C 13, 35%; 12CI2-13CI3, violacein were obtained.
25 0/'
/0'
l3CI2-12C13 , 25/'
/0'
13CI2-13C13 , 15/.
/0 The values of the 13C_13C coupling con-
The 13C atom percentage at Cl3 was also es- stants, 1 lc-c and 21c_o obtained by the feed-
timated to be 42% from an integration of the ing experiments further confirmed the 13C
satellite peaks of H 13 in the 1H NMR spec- NMR assignments of the pyrrolidone moiety.
trum of the labeled violacein 113C13_'H13 = e Taking into consideration the labeled posi-
179Hz). tions, the occurrence of an indole migration is
A similar feeding experiment using labeled apparent (Scheme 1). In conclusion, the ring
DL-[2-13C]tryptophan (13C 90%),8) with 10 mg closure process of the pyrrolidone unit oc-
added to 100 ml of medium, proved the labeled curred by the bond formations of both C3-C3
968 T. HOSIDNO et al.
_~_~"o
-IJ
'CO. A ? I I ~'"
... N
H
A-OH& H
SCHEME 1.
and C2-N -C2 between the two tryptophan side 2) H. C. Lichstein and V. F. van de Sand, J. Infectious
chains, and was accompanied by the l,2-shift Disease, 76, 47 (1945); N. Takahashi, S. Marumo and
N. Otake, "Seiri Kassei Tennenbutsu Kagaku,"
of the indole ring for one molecule of tryp-
Tokyo Daigaku Shuppankai, Tokyo, 1981, p. 308.
tophan (Scheme 1). 3) J. A. Ballantine, R. J. S. Beer, D. J. Crutchley, G. M.
To our knowledge, this is the first report on Dodd and D. R. Palmer, Proc. Chem. Soc., 1958,
rearrangement with the indole 1,2-shift. A new 232; J. Chem. Soc., 1960, 2292.
enzyme of indole transferase may be involved 4) R. D. Demoss and N. R. Evans, J. Bacteriol., 79, 729
in the violacein biosynthesis. Our present in- (1960).
5) "Taisha Mapp," ed. by Nihon Seikagakukai, Tokyo
vestigation is centered upon the more detailed Kagaku Dojin, Tokyo, 1980; "Biosynthesis,"
mechanism(s) for rearrangement and conden- Specialist Periodical Reports, Vok 1 ~ 7, The
sation between the two side chains of trypto- Chemical Society, Burlingon, London, 1972 ~ 1981;
phans, and also upon the elucidation of the K. B. G. Torssell, "Natural Product Chemistry,"
John Wiley & Sons, U.S.A., 1983; Atta-ur-Rahman
biosynthetic intermediates. To elucidate the
and A. Basha, "Biosynthesis of Indole Alkaloids,"
mechanisms, the biosynthetic origin of the
Clarendon Press, Oxford, 1983.
nitrogen, oxygen and hydrogen atoms in the 6) K. Nakanishi, M. Kajiwara and K. Tsutsumi, "Yuki
pyrrolidone moiety must be unabmiguously Kagobutsu Spekutoru Deta Shu," (translation)
disclosed. Further studies on the biosynthesis Kodansha Scientific, Tokyo, 1982, p. 78.
are now in progress. 7) Synthesized from 13C HCHO (13C 97.8%, pur-
chased from MSD, Canada) according to ref. 9).
Acknowledgment. This work was supported by a Optical reaolution was carried out by treating
Grant-in-Aid for Scientific Research (No. 61760106) from aminoacylase to the DL-[3- 13 C] N-acetyltryptophan.
the Ministry of Education, Science and Culture of Japan. The 13C atom percentage was determined by MS
analysis of the tryptophan methyl ester, prepared
REFERENCES AND NOTES with HCI/MeOH.
1) Lecoq de, Biosbaudran, Compt. rend., 94,562 (1882); 8) Purchased from MSD, Canada.
R. D. Demoss, Antibiotics, 2, 77 (1967); "The Merck 9) A. Murray and D. L. Williams, "Organic Syntheses
Index, Tenth Edition," ed. by M. Windholz Merck & with Isotopes," Vol. 1, Interscience, U.S.A., 1958, p.
Co. Inc., U.S.A., 1983, p. 1430. 249.