Agricultural and Biological Chemistry

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Biosynthesis of Violacein: a Novel Rearrangement

in Tryptophan Metabolism with a 1,2-Shift of the
Indole Ring

Tsutomu Hoshino, Tadao Kondo, Takeo Uchiyama & Nagahiro Ogasawara

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

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Agric. Bioi. Chem., 51 (3), 965~968, 1987
Short Communication
Biosynthesis of Violacein: a Novel
Rearrangement in Tryptophan
Metabolism with a 1,2-Shift
of the Indole Ringt
Tsutomu HOSHINO, Tadao KONDO, *
Takeo UCHIYAMA and Nagahiro OGASAWARA
Department of Agricultural Chemistry,
Faculty of Agriculture,
Ikarashi, Niigata 950-21, Japan
*Chemical Instrument Center,
Nagoya University,
Chikusa, Nagoya 464, Japan
Received December 8, 1986
A number of studies have been reported on
the blue pigment, violacein, and its producins
bacterium, Chromobacterium violaceum, since
its discovery in 1882.1) Violacein possesses
antibiotic activities against various micro-
organisms. 2 ) Its' chemical structure was pro-
posed by a series of degradation studies, and
was ascertained by chemical synthesis in
1958. 3Y The structure consists of three units,
i.e., the 5-hydroxyindole, oxindole and 2-pyr-
rolidone moieties. 'Some investigations on the
biosynthesis of violacein have appeared; by
tracer experiments with various 14C-labeled
compounds, it has been demonstrated that L-
R=OH,VIOLACEIN
R=H, DEOXYVIOLACEIN
FIG. 1. Structures of Violacein and Deoxyviolacein.
t Studies on the Biosynthesis .of Violacein. Part I.
965
tryptophan is a biosynthetic precursor with the
involvement of a decarboxylation process. 4 )
However, the biosynthetic pathway and me-
chanism, as well as the origin, of the 2-pyr-
rolidone nucleus are still unknown.
Feeding experiments using [2- 13 C] and [3-
13C]tryptophans have estabiished that the car-
bon skeleton of the pyrrolidone moiety was
constructed by the condensation of the side
chains of two tryptophan molecules, and have
also revealed the interesting phenomenon that
one of the two indole rings migrated to the 2-
position of the tryptophan side chain in the
biosynthetic process. In this communication,
we describe a new biosynthetic rearrangement,
the indole 1,2-shift; such migration reactions
have not been reported so far in either the
metabolism of tryptophan or in the biosyn-
theses of indole alkaloids. 5 ) We also report for
the first time the complete assignments of the
1Hand 13C NMR spectral data of violacein.
All the proton signals of viol acein in DMSO-
d6 (200 MHz) were assigned mainly by ID
spin-decoupling experiments. The 13C signals
directly attached to hydrogens were made clear
by selective proton-decoupling and 2D direct
C-H shift-correlation spectra. Two amide car-
bonyl carbons (CII and C16) were distin-
guished by observing a proton-coupled spec-
trum; long-range coupling 3JCll-H13 (9.6 Hz) was
observed for the 171.5 ppm signal, while the
signal at 170.1 ppm was not definitively split.
The signal at 152.9ppm was determined to be
C6 by comparing the 13C NMR spectrum of
violacein with that of deoxyviolacein; the
signal was absent in the 13C signals of deoxy-
violacein, and the substituent effect of the
hydroxyl group (+29.8 ppm) on the 13C signal
at C6 ofdeoxyviolacein was quite reasonable. 6 )
Its substituent effect was also observed at C5,
C7 and C9 (Table 1).6) The eight unidentified
quaternary carbons could be assigned by the
long-range C-H shift-correlation spectrum
(500 MHz). Maximum polarization transfer
was obtained for JCH = 10 Hz. The assignments
of all the 1Hand 13C NMR signals of violacein
966 T. HOSHINO et al.

TABLE I. 'H AND 13C NMR DATA OF VIOLACEIN AND DEOXYVIOLACEIN IN DMSO-d6

Violacein Deoxyviolacein
Position
13C 'H 13C

I 11.89 (br. s) 12.13 (br. s)

2 8.07 (d, J=2.9)* 129.5 (d) 8.19 (d, J=2.2)* 129.3 (d)
3 105.8 (s) 106.3 (s)
4 125.5 (s) 124.4 (s)
5 7.24 (d, J=2.2) 104.5 (d) 7.85 (m) 119.7 (d)
6 9.35 (br. s) 152.9 (8) around 123.1 (d)
7 6.78 (dd, J=9.2, 2.2) 113.2 (d) } -7.3 (2H, m) 121.5 (d)
8 7.35 (d, J=9.2) 113.4 (d) 7.56 (m) 112.9 (d)
9 131.5 (8) 137.4 (s)
10 10.74 (br. s) 10.83 (br. s)
11 171.5 (s) 171.5 (s)
12 136.9 (s) 136.7 (s)
13 7.55 (d, "":1)** 96.9 (d) 7.68 (s) 97.4 (s)
14 147.5 (s) 147.0 (s)
15 10.64 (br. s) 10.67 (br. s)
16 170.1 (s) 170.1 (s)
17 118.7 (s) 119.5 (8)
18 122.3 (s) 122.2 (s)
19 8.93 (dd, J=1.8, 7.8) 126.3 (d) 8.96 (d, J=7.7) 126.4 (d)
20 6.95 (dt, J= 1.8, 7.8) 120.8 (d) 6.96 (t, J=7.7) 120.9 (d)
21 7.20 (dt, J= 1.8, 7.8) 129.3 (d) 7.22 (t, J=7.7) 129.7 (d)
22 6.82 (dd, J= 1.8,7.8) 108.9 (d) 6.83 (d, J= 7.7) 109.1 (d)
23 141.8 (s) 141.9 (s)

'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
details of these assignments will be reported
elsewhere.
Violacein and deoxyviolacein were pro-
duced during the cultivation of Chromobacte-
rium violaceum JCM 1249 in a nutrient broth
medium (at pH 7) consisting of meat extract
(3 g), polypeptone (5 g), sodium chloride (5 g)
and water (1000 ml), with agitation for 48 hrs
on a rotary shaker at 180 rpm and 25C. Blue
bacterial cells were harvested by centrifugation
and the blue pigments were extracted several
times with methanol. The extract was ad-
sorbed on Amberlite XAD 2, which was then
washed with hot water and 40% aq. methanol,
and subsequently eluted with hot abs. meth-
anol. Separ~tion of violacein and deoxy-
violacein was achieved by column chroma-
tography, using Sephadex LH 20 with an
eluent of methanol; deoxyviolacein was fol-
lowed by violacein. Each blue fraction was
concentrated to a small volume and was
allowed to stand overnight, whereupon blue
crystalline pigments gradually separated out.
The addition of 25 mg of tryptophan to
1000 ml of medium increased the pigment pro-
duction by about l.5 fold.
L-[3-13C]tryptophan (13C 94%, 5 mgf) was
administered to a 300 ml erlenmeyer flask con-
taining 100 ml of medium, which was then
cultured under the conditions previously men-
tioned (yield, 20mg of viol ace in and 3.5mg of
deoxyviolacein from six flasks). High level
enrichment of the i3C atoms was observed at
both C12 and C13 in the proton-noise de-
coupled 13C NMR of violacein (Fig. 2). Satel-
 Biosynthesis of Violacein 967

12 13

1
J c_c (12-13)

(A) [3_ 13 CJ Trp fed = 55.1 Hz

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

200 175 150 125 100 ppm

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
contiguous 13C atoms were also observed as a
result of the high incorporation rate of labeled
tryptophan. These findings suggest that the
carbon skeleton of the pyrrolidone ring was
formed by the condensation of the side chains
of two tryptophan molecules. Anal;:ses of El-
Ms and the ratio between the satellite peaks
and the central peak inthe l3C NMR spectrum
are indicative of the following isotopic dis-
tribution: 12C 12-12 C 13, 35%; 12CI2-13CI3,
25 0/'
/0'
l3CI2-12C13 , 25/'
/0'
13CI2-13C13 , 15/.
The 13C atom percentage at Cl3 was also es-
timated to be 42% from an integration of the
satellite peaks of H 13 in the 1H NMR spec-
trum of the labeled violacein 113C13_'H13 =
179Hz).
A similar feeding experiment using labeled
DL-[2-13C]tryptophan (13C 90%),8) with 10 mg
added to 100 ml of medium, proved the labeled
positions of both Cll and Cl4 as shown in
Fig. 2 (note the enriched signals and the
satellite peaks arising from two-bond 13C_13C
coupling via a nitrogen atom). The 13C atom
percentages at the labeled positions were close
to those of violacein produced by feeding with
[3-13C]tryptophan.
In the case of deoxyviolacein, identical in-
corporation patterns and almost the same
incorporation percentage of the isotope as
violacein were obtained.
/0 The values of the 13C_13C coupling con-
stants, 1 lc-c and 21c_o obtained by the feed-
ing experiments further confirmed the 13C
NMR assignments of the pyrrolidone moiety.
e Taking into consideration the labeled posi-
tions, the occurrence of an indole migration is
apparent (Scheme 1). In conclusion, the ring
closure process of the pyrrolidone unit oc-
curred by the bond formations of both C3-C3
968 T. HOSIDNO
_~_~"o
-IJ
'CO.
SCHEME
and C2-N -C2 between the two tryptophan side
chains, and was accompanied by the l,2-shift
of the indole ring for one molecule of tryp-
tophan (Scheme 1).
To our knowledge, this is the first report on
rearrangement with the indole 1,2-shift. A new
enzyme of indole transferase may be involved
in the violacein biosynthesis. Our present in-
vestigation is centered upon the more detailed
mechanism(s) for rearrangement and conden-
sation between the two side chains of trypto-
phans, and also upon the elucidation of the
biosynthetic intermediates. To elucidate the
mechanisms, the biosynthetic origin of the
nitrogen, oxygen and hydrogen atoms in the
pyrrolidone moiety must be unabmiguously
disclosed. Further studies on the biosynthesis
are now in progress.
Acknowledgment. This work was supported by a
Grant-in-Aid for Scientific Research (No. 61760106) from
the Ministry of Education, Science and Culture of Japan.
REFERENCES AND NOTES
1) Lecoq de, Biosbaudran, Compt. rend., 94,562 (1882);
R. D. Demoss, Antibiotics, 2, 77 (1967); "The Merck
Index, Tenth Edition," ed. by M. Windholz Merck &
Co. Inc., U.S.A., 1983, p. 1430.
et al.
A ? I I ~'"
... N
H
A-OH& H
1.
2) H. C. Lichstein and V. F. van de Sand, J. Infectious
Disease, 76, 47 (1945); N. Takahashi, S. Marumo and
N. Otake, "Seiri Kassei Tennenbutsu Kagaku,"
Tokyo Daigaku Shuppankai, Tokyo, 1981, p. 308.
3) J. A. Ballantine, R. J. S. Beer, D. J. Crutchley, G. M.
Dodd and D. R. Palmer, Proc. Chem. Soc., 1958,
232; J. Chem. Soc., 1960, 2292.
4) R. D. Demoss and N. R. Evans, J. Bacteriol., 79, 729
(1960).
5) "Taisha Mapp," ed. by Nihon Seikagakukai, Tokyo
Kagaku Dojin, Tokyo, 1980; "Biosynthesis,"
Specialist Periodical Reports, Vok 1 ~ 7, The
Chemical Society, Burlingon, London, 1972 ~ 1981;
K. B. G. Torssell, "Natural Product Chemistry,"
John Wiley & Sons, U.S.A., 1983; Atta-ur-Rahman
and A. Basha, "Biosynthesis of Indole Alkaloids,"
Clarendon Press, Oxford, 1983.
6) K. Nakanishi, M. Kajiwara and K. Tsutsumi, "Yuki
Kagobutsu Spekutoru Deta Shu," (translation)
Kodansha Scientific, Tokyo, 1982, p. 78.
7) Synthesized from 13C HCHO (13C 97.8%, pur-
chased from MSD, Canada) according to ref. 9).
Optical reaolution was carried out by treating
aminoacylase to the DL-[3- 13 C] N-acetyltryptophan.
The 13C atom percentage was determined by MS
analysis of the tryptophan methyl ester, prepared
with HCI/MeOH.
8) Purchased from MSD, Canada.
9) A. Murray and D. L. Williams, "Organic Syntheses
with Isotopes," Vol. 1, Interscience, U.S.A., 1958, p.
249.