A novel type of luciferin from the siberian luminous earthworm fridericia heliota: Structure elucidation by spectral studies and total synthesis

Article abstract of DOI:10.1002/anie.201400529

The structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota is reported. This luciferin is a key component of a novel ATP-dependent bioluminescence system. UV, fluorescence, NMR, and HRMS spectroscopy studies were performed on 0.005 mg of the isolated substance and revealed four isomeric structures that conform to spectral data. These isomers were chemically synthesized and one of them was found to produce light when reacted with a protein extract from F. heliota. The novel luciferin was found to have an unusual extensively modified peptidic nature, thus implying an unprecedented mechanism of action. Feeling blue: UV, fluorescence, NMR, and HRMS spectroscopy studies were used to elucidate the structure of the luciferin from the recently discovered luminous earthworm Fridericia heliota. The luciferin, which is part of a novel ATP-dependent bioluminescent system, was found to be a peptide formed from oxalic acid, lysine, a modified tyrosine residue, and γ-aminobutyric acid.

Full text of DOI:10.1002/anie.201400529

Angewandte
Chemie
DOI: 10.1002/anie.201400529
Bioluminescence
A Novel Type of Luciferin from the Siberian Luminous Earthworm
Fridericia heliota: Structure Elucidation by Spectral Studies and Total
Synthesis**
Valentin N. Petushkov, Maxim A. Dubinnyi, Aleksandra S. Tsarkova, Natalja S. Rodionova,
Mikhail S. Baranov, Vadim S. Kublitski, Osamu Shimomura, and Ilia V. Yampolsky*
Abstract: The structure elucidation and synthesis of the
luciferin from the recently discovered luminous earthworm
Fridericia heliota is reported. This luciferin is a key component
of a novel ATP-dependent bioluminescence system. UV,
fluorescence, NMR, and HRMS spectroscopy studies were
performed on 0.005 mg of the isolated substance and revealed
four isomeric structures that conform to spectral data. These
isomers were chemically synthesized and one of them was
found to produce light when reacted with a protein extract from
F. heliota. The novel luciferin was found to have an unusual
extensively modified peptidic nature, thus implying an unpre-
cedented mechanism of action.
production is generated by the oxidation of a small organic
molecule, luciferin, facilitated by a specific enzyme, lucifer-
ase. To date, the structures of only seven natural luciferins are
known (Figure S1 in the Supporting Information).[1] The last
structural characterization of a novel luciferin, from dino-
flagellates, dates back 25 years.[2]
Recently, a novel bioluminescent species was discovered
in Siberia.[3] Fridericia heliota is a small (ca. 15 mm in length,
0.5 mm in diameter, and ca. 2 mg in weight) white-yellowish
oligochaete worm that inhabits forest soils and emits blue
light (with a luminescence maximum at 478 nm) when
mechanically stimulated. The luminescence of F. heliota is
located in the region of the epidermal cells (Figure 3B).
The general concept of the common nature of lumines-
cence in earthworms was based on the results of comparative
studies of the physiology and biochemistry of 12 species
belonging to 6 genera (Diplocardia, Diplotrema, Fletchero-
drilus, Octochaetus, Pontodrilus, and Spenceriella).[4] All of
these bioluminescent oligochaetes secrete a luminescent
liquid containing coelomic cells, in the granules of which the
luminescence is localized. Bioluminescence in oligochaetes is
characterized by a common feature: the involvement of
hydrogen peroxide. The luciferin of Diplocardia longa, N-
isovaleryl-3-amino-propanal, serves as a substrate for the
luciferases of all bioluminescent earthworms. In addition, D.
longa luciferase is active in cross reactions with luciferins
from other worms.[5]
The bioluminescence of potworms (of the family Enchy-
traeidae) has been known since 1838, although in lesser detail
than that of megadriles, and it is confined to the genera
Henlea and Fridericia.[6]
We demonstrated the light-production mechanism of F.
heliota to be unique, since cross-reaction experiments with the
luciferase or luciferin from Fridericia with luciferins and
luciferases from other organisms were all negative. We found
five components to be essential for F. heliota luminescence:
Fridericia luciferase, Fridericia luciferin, ATP, Mg2+, and
atmospheric oxygen.[7,8]
B
ioluminescence is a fascinating phenomenon in which
visible light is emitted by living organisms. Hundreds of
species of bioluminescent animals, fungi, protists, and bacteria
are known, and there are estimated to be 30 different
chemical mechanisms underlying the generation of “cold
light”.[1] In all known cases, the energy required for light
[*] V. N. Petushkov, N. S. Rodionova, O. Shimomura
Laboratory of Bioluminescent Biotechnologies
Institute of Fundamental Biology and Biotechnology
Siberian Federal University
pr. Svobodnyi, 79, Krasnoyarsk 660041 (Russia)
V. N. Petushkov, N. S. Rodionova
Laboratory of Photobiology, Institute of Biophysics
Siberian Branch of the Russian Academy of Sciences
Akademgorodok, Krasnoyarsk 660036 (Russia)
M. A. Dubinnyi, A. S. Tsarkova, M. S. Baranov, V. S. Kublitski,
I. V. Yampolsky
Institute of Bioorganic Chemistry, Russian Academy of Sciences
Miklukho-Maklaya 16/10, Moscow 117997 (Russia)
E-mail: ivyamp@ibch.ru
O. Shimomura
Marine Biological Laboratory
Woods Hole, MA 02543 (USA)
[**] We thank Dr. Alexander O. Chizhov for recording mass spectra and
Dr. K. S. Mineev for NMR analysis of synthetic intermediates. We
acknowledge support from the Program of the Government of the
Russian Federation “Measures to attract leading scientists to
Russian educational institutions” (grant no. 11. G34.31.0058), the
programs MCB RAS, President of the Russian Federation “Leading
science school” (grant 3951.2012.4) and the Russian Foundation for
Basic Research (grant 14-03-01015). B.M.S. was supported by
a stipend from the Program of the President of the Russian
Federation.
The isolation and structural characterization of Fridericia
luciferin has been seriously hindered by the scarcity of
earthworm biomass (manual harvesting gave about 30 g/
year), and the low content of luciferin (ca. 0.1 mg per gram of
wet biomass).[9] In the course of our extensive efforts aimed at
purification of Fridericia luciferin, we isolated CompX,
a component of Fridericia biomass that is similar to luciferin
by chromatographic and UV spectral behavior.[10] Spectral
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201400529.
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studies supported by total synthesis
revealed CompX to be an unusual
derivative of tyrosine (Figure 1).
The total amount of luciferin
obtained from 90 g of biomass was
0.005 mg, and this small amount
allowed us to obtain only the 1H,
COSY and partial 13C-HSQC NMR
spectra (Figure S4–S6). These data
revealed the following three frag-
ments of luciferin structure: substi-
tuted CompX, lysine, and g-amino-
butyric acid (GABA). The scarcity of
luciferin did not allow us to obtain
1D 13C and HMBC spectra, which
would have revealed the connectivity
Figure 1. CompX, a struc-
tural analogue of Frider-
icia luciferin found in Fri-
dericia biomass.
of these three fragments and the presence of non-hydro-
genated carbon atoms. Obviously, the most plausible way for
these fragments to form a stable compound while leaving all
À
of their C H bonds intact (a requirement imposed by
available NMR data) is the formation of peptide bonds
between some of their four carboxylic acid and three amino
groups. Therefore, we performed an 1H NMR titration of
luciferin in the pH range 3.1–7.5 in order to discriminate
between the free carboxylic groups and those forming peptide
bonds with the amino groups of the lysine and GABA
residues. The pH dependence of the chemical shifts of the
protons adjacent to titratable carboxy groups indicated that
the carboxy groups of lysine and GABA are free, whereas the
two carboxy groups of the CompX moiety are probably
involved in peptide bonds (Figure S7).
The HRMS spectra of luciferin showed a molecular ion
with m/z = 524.1851, for which the closest molecular formula
is C23H30N3O11+. The difference between this formula and
(CompX + Lysine + GABAÀ2H2O) is C2O3. In this calcula-
tion, the loss of two water molecules is assumed to result from
the formation of two peptide bonds between the three
specified fragments. Furthermore, the MS spectra showed
two abundant peaks corresponding to the loss of CO2 and of
both CO2 and CO, but not of CO alone (Figure S8). Taken
together, these data suggest a monosubstituted oxalic acid
residue to be a missing structural fragment of luciferin.
Four isomeric structures (1–4; Figure 2) were consistent
with the NMR and mass spectroscopy data summarized
above. These isomers differ only by the order of the peptide
bonds connecting the four residues identified as the building
blocks of Fridericia luciferin: CompX, lysine, GABA, and
oxalate. Derivatives of l-lysine and racemization-preventing
conditions were used throughout all of the syntheses.
Figure 2. A) Structures of the synthetic isomeric peptides 1–4. Only
1 produced light when mixed with Fridericia luciferase. B) A compar-
ison of selected fragments of the 13C-HSQC NMR spectra of Fridericia
luciferin and compounds 1–4 (D2O, 308C, pH 5.0). The colors of the
peaks correspond to the compound colors shown in (A); the peaks for
natural luciferin are shown in black.
We explored the ability of synthetic compounds 1–4 to
produce light upon addition to the crude Fridericia luciferase
in the presence of ATP and MgSO4. Only synthetic compound
1 produced luminescence under these conditions, with
We synthesized the isomeric peptides 1–4 and compared
their NMR spectra with those of the natural sample (Table S1
in the Supporting Information). The synthesis started from
CompX monomethyl ester[10] and utilized common peptide
chemistry methods (Schemes S1–S4 in the Supporting Infor-
mation). All of synthetic compounds gave similar NMR
a
luminescence spectrum and intensity-concentration
dependence identical to those of the natural luciferin
(Figure 3 and Figure S3).
In summary, we report the structure of a novel luciferin,
which represents a key component of a novel ATP-dependent
bioluminescence system from the Siberian earthworm Frider-
icia heliota. This luciferin probably represents a fundamentally
spectra in D2O at pH 5.0. However, only 1 showed 1H and 13
C
chemical shifts completely matching those of the natural
luciferin (Figure 2B, Table S1).
2
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moiety is found in F. heliota biomass. One such peptide (a
tyrosine–CompX–lysine derivative designated AsLn2) was
described in our recent paper.[10b] The structures of other
members of this family and their role in luciferin metabolism
are presently under investigation. Our further efforts will be
focused on the structural characterization of luciferin biosyn-
thetic precursors and oxyluciferin, evaluation of the role of
ATP, and on sequencing and cloning Fridericia luciferase.
Received: January 17, 2014
Revised: February 18, 2014
Published online: && &&, &&&&
Keywords: bioluminescence · luciferin · natural products ·
.
NMR spectroscopy · total synthesis
[1] O. Shimomura, Bioluminescence: Chemical Principles and
Methods, World Scientific Publishing, Singapore, 2006.
[2] H. Nakamura, Y. Kishi, O. Shimomura, D. Morse, J. W. Hastings,
J. Am. Chem. Soc. 1989, 111, 7607 – 7611.
Figure 3. A) The structure of Fridericia luciferin. B) The biolumines-
cence of Fridericia heliota. The photograph is courtesy of Alexander
Semenov (White Sea Biological Station, Biology Department of Lomo-
nosov Moscow State University). C) The luminescence of synthetic
Fridericia luciferin. D) A comparison of the in vivo bioluminescence
spectra of the worms (yellow dots) with the in vitro bioluminescence
spectra of natural (white line) and synthetic (blue line) samples of the
luciferin.
[3] E. Rota, N. T. Zalesskaja, N. S. Rodionova, V. N. Petushkov, J.
Zool. 2003, 260, 291 – 299.
[4] J. E. Wampler, B. G. M. Jamieson, Comp. Biochem. Physiol. Part
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[5] H. Ohtsuka, N. G. Rudie, J. E. Wampler, Biochemistry 1976, 15,
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[6] “Lights on the ground: A historical survey of light production in
the Oligochaeta”: E. Rota in Bioluminescence in Focus—A
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[7] V. N. Petushkov, N. S. Rodionova, V. S. Bondar, Dokl. Biochem.
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[10] a) V. N. Petushkov, A. S. Tsarkova, M. A. Dubinnyi, N. S.
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novel chemical mechanism of luminescence, which will be
evaluated in the near future. Presumably, an oxalate moiety is
oxidized in the course of the luminescence reaction while
a fluorescent CompX moiety serves as a light emitter, in
a manner similar to the chemistry underlying the “glow stick”
chemiluminescence toys. The role the of the CompX moiety
as the light emitter is supported by the close similarity of
luciferin fluorescence emission spectrum to the biolumines-
cence spectrum of Fridericia heliota (lmax 466 and 480 nm,
respectively).10 An interesting question concerns the biosyn-
thetic route that leads to Fridericia luciferin. In a recent study
by Clardy et al., a carbonylated analogue of tyrosine similar
to the CompX fragment was identified through genome
screening for the members of the ATP-grasp enzyme family.11
Considering the nonribosomal peptidic nature of the novel
luciferin, its biosynthesis might utilize the enzymes of this
family. A set of unusual peptides containing the CompX
[11] L. C. Blasiak, J. Clardy, J. Am. Chem. Soc. 2010, 132, 926 – 927.
Angew. Chem. Int. Ed. 2014, 53, 1 – 4
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angewandte
Communications
Communications
Bioluminescence
V. N. Petushkov, M. A. Dubinnyi,
A. S. Tsarkova, N. S. Rodionova,
M. S. Baranov, V. S. Kublitski,
O. Shimomura,
I. V. Yampolsky*
&&&& — &&&&
A Novel Type of Luciferin from the
Siberian Luminous Earthworm Fridericia
heliota: Structure Elucidation by Spectral
Studies and Total Synthesis
Feeling blue: UV, fluorescence, NMR, and
HRMS spectroscopy studies were used to
elucidate the structure of the luciferin
from the recently discovered luminous
earthworm Fridericia heliota. The luciferin,
which is part of a novel ATP-dependent
bioluminescent system, was found to be
a peptide formed from oxalic acid, lysine,
a modified tyrosine residue, and g-ami-
nobutyric acid.
4
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 4
These are not the final page numbers!