2005-09-10467B, Hiroaki Kato
Supplementary Discussion
Sequence analysis
The click beetle, Pyrophorus plagiophthalamus (Ppl), is known to possess four
types of luciferases distinguished by their luminescence color: green (546 nm),
yellow-green (560 nm), yellow (578 nm) and orange (593 nm)1. The amino-acid
sequences of the beetle luciferases are 95 to 99% identical with each other, but are only
ca. 50% identical with those of the firefly luciferases.
Interestingly, the Ppl luciferases
that emit green (PplGR) and yellow-green light (PplYG) possess an Ile residue at the
position corresponding to Ile288 of Lcr luciferase, but this position is substituted with a
Val residue in the Ppl luciferases that emit light with longer wavelengths: yellow
(PplYE) and orange (PplOR) (Fig. S2). We therefore believe that the translocation of
the side chain of Ile288 generally controls the bioluminescence spectra by affecting the
nature of the microenvironment for the excited state of oxyluciferin. The change in the
hydrogen bonding at Ser286 might serve as a molecular switch that triggers the
movement of Ile288.
A luciferase from a railroad-worm Phrixothrix_vivianii (PvGR)2 emits green light
(549 nm), but has a Val residue at the position corresponding to Ile288 of Lcr luciferase.
In PvGR, however, two additional key residues corresponding to Ser286 and Ser349 of
firefly luciferases are mutated to Thr and Cys residues, respectively.
1
The overall
2005-09-10467B, Hiroaki Kato
effects of these mutations on bioluminescence color are not fully understood at the
current level of our structural study, but do not exclude the possibility of general and
integral role of Ile288 in the firefly luciferases.
We therefore conclude that the
conformational change involving the movement of Ile288 is a common, but not the sole
catalytic machinery that is used widely by insect luciferases to control their
bioluminescence spectra.
2
2005-09-10467B, Hiroaki Kato
Supplementary Methods
Synthesis of 5’-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA)
In the molecular design of DLSA, we used dehydroluciferin, rather than luciferin itself,
as the luciferyl moiety of the ligand to mimic the growing sp2-character at C-4 in the
thiazoline ring predicted in the subsequent oxidation step of the luciferyl adenylate
intermediate by molecular oxygen.
Furthermore, the planar thiazole ring is analogous
to oxyluciferin in a dianionic enolate form as a putative emitter for the yellow-green
bioluminescence3, thereby allowing DLSA to mimic the late transition state for the
activation of luciferin and/or the excited state of oxyluciferin. During the course of
our study, however, the same molecule was synthesized as an inhibitor of Ppy
luciferase4. The followings are our synthetic route.
General methods.
Compounds were purified by flash column chromatography on silica gel 60 (230–400
mesh,
Merck,
No.
9385)
or
by
reversed-phase
medium-pressure
column
chromatography on an ULTRA PACK™ octadecyl silica gel column (ODS-S-50B, 50
1
mm, 120 Å, Yamazen Co., Osaka, Japan).
H and 13C NMR spectra were recorded on
a Varian VXR-200 (200 MHz) or a JEOL JNM-AL400 (400 MHz) spectrometer using
tetramethylsilane (for CDCl3 and DMSO-d6) or 3-(trimethylsilyl)propanesulfonic acid
3
2005-09-10467B, Hiroaki Kato
sodium salt (for D2O) as an internal standard. Chemical shifts were recorded in ppm
with the internal standard set at H = 0.00. Elemental analyses were performed on a
Yanaco MT-5 system.
Mass spectra were obtained on a JEOL JMS700 spectrometer.
Dehydroluciferin pentafluorophenyl ester (Compound 1)
A
solution
of
2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic
acid
(dehydroluciferin, DL)5,6 (111 mg, 0.4 mmol) and EDC (96 mg, 0.5 mmol) in pyridine
(20 ml) was mixed with pentafluorophenol (92 mg, 0.5 mmol), and the mixture was
stirred at ambient temperature for 16 h. Pyridine was removed by evaporation, and the
residue was taken up in a mixture of AcOEt and MeOH (9:1). The solution was
filtered, washed with 0.1 M HCl, and concentrated in vacuo to leave a brown oil. The
residual oil was purified by flash column chromatography on silica gel (gradient of 0 to
100% AcOEt in hexane) to give Compound 1 as a yellow solid (250 mg, 70% purity)
(84% yield).
1
H-NMR (200 MHz, DMSO-d6) H: 9.23 (s, 1H, thiazole H-5), 7.98 (d,
1H, J = 8.8 Hz, benzothiazole H-4), 7.50 (d, 1H, J = 2.4 Hz, H-7), 7.09 (dd, 1H, J = 9
and 2.4 Hz, H-5).
2’,3’-O-Isopropylidene-5’-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (2)
A solution of DBU (91 mg, 0.6 mmol) was added at ambient temperature to a solution
4
2005-09-10467B, Hiroaki Kato
of 2’, 3’-O-isopropylidene-5’-sulfamoyladenosine7 (160 mg, 0.41 mmol) in dry DMF
(14 ml). After 10 min, a solution of Compound 1 (180 mg, 0.41 mmol) in dry DMF (1
ml) was added.
The mixture was stirred under a nitrogen atmosphere for 16 h.
Pyridine (1 ml) was added, and the mixture was stirred for additional 4 h.
The reaction
mixture was concentrated in vacuo, and the residual oil was purified by flash column
chromatography on silica gel (gradient of 5 to 30% MeOH in AcOEt) to give
Compound 2 as a bright yellow solid (55 mg, 21%).
1
H-NMR (200 MHz, DMSO-d6)
H: 8.44 (s, 1H, thiazole H-5), 8.23 (s, 1H, adenine H-8), 8.13 (s, 1H, adenine H-2), 7.90
(d, 1H, J = 9.0 Hz, benzothiazole H-4), 7.48 (d, 1H, J = 2.2 Hz, H-7), 7.3 (br s, 2H,
NH2), 7.07 (dd, 1H, J = 9.0 and 2.2 Hz, H-5), 6.19 (d, 1H, J = 3.0 Hz, ribose 1’-H), 5.41
(dd, 1H, J = 6.0 and 3.0 Hz, 2’-H), 5.08 (dd, 1H, J = 6.2 and 2.2 Hz, 3’-H), 4.5 (m, 1H,
4’-H), 4.1 (m, 2H, 5’-H).
5’-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA)
Compound 2 (55 mg, 0.09 mmol) was dissolved in a mixture of trifluoroacetic acid and
water (2:1 v/v, 3 ml). The mixture was stirred for 3 h and was evaporated.
trifluoroacetic acid was removed by azeotrope with ethanol and toluene.
A trace of
The residual
oil was taken up in water, filtered, and purified by a reversed-phase column
chromatography on a Dianion HP20SS resin (linear gradient of 0 to 30% THF in water).
5
2005-09-10467B, Hiroaki Kato
The fractions containing DLSA (Abs. 254 nm) was evaporated and lyophilized from
water to afford DLSA as a bright yellow light solid (9 mg, 17%).
1
H-NMR (400 MHz,
CD3OD) H: 8.51 (s, 1H, thiazole H-5), 8.32 (s, 1H, adenine H-8), 8.15 (s, 1H, adenine
H-2), 7.83 (d, 1H, J = 8.9 Hz, benzothiazole H-4), 7.32 (d, 1H, J = 2.4 Hz, H-7), 7.02
(dd, 1H, J = 8.9 and 2.4 Hz, H-5), 6.08 (d, 1H, J = 5.5 Hz, ribose 1’-H), 4.72 (t, 1H, J =
5.3 Hz, 2’-H), 4.4-4.5 (m, 3H, 3’ and 5’-H), 4.3 (m, 1H, 4’-H).
HRMS (FAB,
4-nitrobenzyl alcohol): Calcd. for C21H19N8O8S3 (M+H+), 607.0491; found, 607.0521;
Calcd. for C21H18N8O8S3Na (M+Na+), 629.0307; found, 629.0303.
DNA Techniques
pTM18 contains the cDNA of the thermostable mutant luciferase (T217I) from the
firefly Luciola cruciata, which produces the same yellow-green light as the wild-type
enzyme does; pGLf37-CM19 contains the cDNA of the mutant luciferase (S286N),
which displays an orange-shifted bioluminescence emission spectra.
Nco I and BamH
I sites were introduced by the polymerase chain reaction to 5’ and 3’ ends, respectively,
of a 1.6-kb fragment that included the T217I cDNA; the ATG sequence in the restriction
site of Nco I (CCATGG) was matched with the initiation codon of T217I luciferase.
For construction of a T217I luciferase with the three N-terminal amino acids deleted,
the restriction sequence for Nco I site was introduced in the region from 8 to 13
6
2005-09-10467B, Hiroaki Kato
nucleotides downstream of the initiation codon. The synthetic mutagenic primers used
were
as
follows
(the
restriction
sites
are
underlined):
forward
primer,
5’-CATGCCATGGAAAACATGGAAAACGATGAA; forward primer for the deletion
mutant,
5’-CCCGCCATGGAAAACGATGAAAATATTGTA;
5’-AAGGATCCTCACATCTTAGCAACTGG.
reverse
primer,
The resultant DNA fragments were
digested with Nco I and BamH I, and ligated to pTV118N (Takara, Shiga, Japan)
digested with the same enzymes to yield pTVLt and pTVLt, respectively (t:
thermostable).
A 0.85-kb Hpa I (the restriction site was located at nucleotide position
789 to 794 from the initiation codon)–BamH I fragment isolated from pTVLt was
replaced with a fragment derived from pGLf37-CM1 digested with the same enzymes to
construct pTVLtS286N.
method
with
Mutants, I288A and I288V, were constructed by PCR
minor
modification.
Primers
used
were
5'-AAACAAGGTCGGTACCAGAGCAACACTTGTACATTT-3',
5'-CAAGATTATAAATGTACTAGTGTTGTTCTTGTACCGACCTTG-3', and primers
used when constructing pTVLt, and pTVLt was used as template.
The
nucleotide sequences of all of the plasmid inserts were confirmed with an Applied
Biosystems 310 DNA sequencer (Foster City, CA).
Protein Expression and Purification
7
2005-09-10467B, Hiroaki Kato
Wild-type (T217I) and mutant luciferases were produced in Escherichia coli HB101
cells. The recombinant E. coli cells producing the enzymes were grown aerobically at
30˚C for 18 h in LB medium containing 0.1 mg/ml ampicillin. The cells harvested
from a 4-L culture were disrupted by sonication. The cell lysate was subjected to
centrifugation at 75,600 x g for 20 min to remove cellular debris.
The resulting
cell-free extract was brought to 30% saturation of ammonium sulfate, and the
precipitated protein was removed by centrifugation.
The soluble supernatant was
dialyzed against buffer 1A (10 mM potassium phosphate (pH 6.8), 5% (w/v) glycerol, 1
mM dithiothreitol (DTT), and 1 mM ethylenediaminetetraacetic acid (EDTA)). The
enzyme was first purified at room temperature with an ÄKTA explorer 10S system
(Amersham Biosciences) and a HiLoad 16/10 SP Sepharose FF cation exchanger
column (column volume: 20 ml) employing a linear gradient of the buffers 1A and 1B
(500 mM potassium phosphate (pH 6.8), 5% (w/v) glycerol, 1 mM DTT, and 1 mM
EDTA); sample injection: 0% B, 1 CV; 0–18% B, 6 CV; 100% B, 1 CV (flow rate: 3
ml/min).
The active fractions were pooled, dialyzed against buffer 2A (10 mM
potassium phosphate (pH 6.8), 5% (w/v) glycerol, and 1 mM DTT), and applied to a
Bio-Scale CHT5-I hydroxyapatite column (column volume: 5 ml) connected to the
ÄKTA system. The enzyme was eluted employing a linear gradient of the buffers 2A
and 2B (500 mM potassium phosphate (pH 6.8), 5% (w/v) glycerol, and 1 mM DTT);
8
2005-09-10467B, Hiroaki Kato
sample injection: 0% B, 3 CV; 0–40% B, 16 CV; 100% B, 1 CV (flow rate: 4 ml/min).
The active fractions were concentrated, dialyzed against 20 mM Tris-HCl (pH 8.0), 10%
(w/v) glycerol, and 2 mM 2-mercaptoethanol, and used as a purified preparation of the
enzyme.
Seeding techniques for crystallization
In order to produce a seeding solution, the clusters of crystals were placed in 60 l of
the reservoir solution and crushed. Tenfold serial dilutions of the crushed crystals
were made in the reservoir solution.
Hanging drops were formed with the enzyme
solution and the diluted seeding solution.
Several single crystals were obtained from
approximately 104-fold dilutions of seeds after 2–3 days.
Measurement of bioluminescence emission spectra
Emission spectra were recorded by multichannel spectrophotometer MCPD-7000
(Otsuka Electronics, Japan).
Substrate solution (500 L) containing 100 mM
HEPES-Na (pH 7.8), 0.6 mM luciferin, 3.2 mM ATP, and 10 mM MgSO4 was mixed
with the wild-type or mutant (S286N, I288V, or I288A) luciferases solution (500 L) in
a quartz cell.
The concentration of the wild-type and mutant are 70 and 140 g/ml,
respectively.
9
2005-09-10467B, Hiroaki Kato
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Wood, K. V., Lam, Y. A., Seliger, H. H. & McElroy, W. D. Complementary DNA
coding click beetle luciferases can elicit bioluminescence of different colors.
Science 244, 700-2 (1989).
Viviani, V. R., Bechara, E. J. & Ohmiya, Y. Cloning, sequence analysis, and
expression of active Phrixothrix railroad-worms luciferases: relationship
between bioluminescence spectra and primary structures. Biochemistry 38,
8271-9 (1999).
White, E. H., Rapaport, E., Hopkins, T. A. & Seliger, H. H. Chemi- and
bioluminescence of firefly luciferin. J Am Chem Soc 91, 2178-80 (1969).
Branchini, B. R., Murtiashaw, M. H., Carmody, J. N., Mygatt, E. E. &
Southworth, T. L. Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a
stable and potent inhibitor of firefly luciferase. Bioorg Med Chem Lett 15,
3860-4 (2005).
White, E. H., McCapra, F. & Field, G. F. The structure and synthesis of firefly
luciferin. 85, 337-343 (1963).
Bowie, L. Synthesis of firefly luciferin and structural analogs. Methods Enzymol
57, 15-28 (1978).
Heacock, D., Forsyth, C. J., Shiba, K. & Musier.-Forsyth, K. Synthesis and
Aminoacyl-tRNA Synthetase Inhibitory Activity of Prolyl Adenylate Analogs.
Bioorg. Chem. 24, 273-289 (1996).
Kajiyama, N. & Nakano, E. Thermostabilization of firefly luciferase by a single
amino acid substitution at position 217. Biochemistry 32, 13795-9 (1993).
Kajiyama, N. & Nakano, E. Isolation and characterization of mutants of firefly
luciferase which produce different colors of light. Protein Eng 4, 691-3 (1991).
10
2005-09-10467B, Hiroaki Kato
Supplementary Figure Legends
Figure S1
a, Stereo view of the luciferin binding site of LcrLuc (WT) in complex
with DLSA.
The polar interactions are shown as dashed lines. The
sulfamoyladenosine moiety of DLSA is omitted from the figure for clarity.
b,
Superposition of the structures of LcrLuc (WT):MgATP (light blue) and LcrLuc
(WT):AMP:oxyluciferin (white) complexes.
dashed lines.
The polar interactions are shown as
The - and - phosphates of ATP are not visible.
c,
Superposition of the LcrLuc (WT) : DLSA (green) and LcrLuc (S286N) : DLSA
(pink) complexes.
The polar interactions are shown as dashed lines.
Figure S2 Sequence alignment of firefly and click beetle luciferases.
Firefly
luciferases: Lcr, Luciola cruciata; Lla, Luciola lateralis; Lmi, Luciola mingrelica;
Hpa, Hotaria parvula; Pmi, Pyrocoelia miyako; Pru, Pyrocoelia rufa; Lno,
Lampyris noctiluca; Ppy, Photinus pyralis; Ppe, Photuris pennsylvanica.
Click
beetle luciferases: Ppl, Pyrophorus plagiophthalamus; Pyt, Pyrearinus
termitilluminans.
The four Ppl luciferases with differently colored emission
spectra are indicated as PplYE (yellow), PplOR (orange), PplYG (yellow-green),
and PplGR (green). The secondary structures involved in the luciferin-binding
site are drawn in red.
The position of Ile 288 and the conserved key residues
11
2005-09-10467B, Hiroaki Kato
are highlighted in black and red, respectively.
Figure S3 Synthesis of 5’ -O-[N-(dehydroluciferyl)-sulfamoyl] adenosine
(DLSA). (i) pentafluorophenol, EDC, pyridine, 25˚C, 16 hr, 84%;
(ii) 2’,
3’-O-isopropylidene-5’-sulfamoyladenosine, 1, DBU, DMF, 25˚C, 20 hr, 21%;
(iii) TFA - water (2:1), 25˚C, 3 hr, Dianion HP20SS (0 to 30% water in THF),
17%.
12
2005-09-10467B, Hiroaki Kato
Supplementary Movie Legends
Movie S1 Movements involved in the luminescence reaction of wild-type
luciferase. The movie is composed of 4 steps; (1) before reaction (MgATP
complex, The -, and - phosphates of ATP are not visible.), (2) intermediate
(mimicked by DLSA complex), (3) excited state (model) and (4) after reaction
(AMP:oxyluciferin complex). Excited state is a speculative model predicted by
DLSA and AMP:oxyluciferin complexes. The excited oxyluciferin is drawn in
green.
All ligands are shown in ball and stick model. Ile288 (blue) and the
other amino acid residues are drawn in space-filing and stick models,
respectively.
The polar interactions are shown as dashed lines.
Movie S2 Movements involved in the luminescence reaction of S286N luciferase.
The movie is composed of 4 steps; (1) before reaction (calculated with the
wild-type MgATP complex), (2) intermediate (mimicked by DLSA complex), (3)
excited state (model) and (4) after reaction (AMP:oxyluciferin complex;
unpublished data). Excitation state is a speculative model predicted by DLSA
and AMP:oxyluciferin complexes.
The excited oxyluciferin is drawn in red.
The other settings are the same as in Movie S1.
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2005-09-10467B, Hiroaki Kato
Supplementary Table
Table S1 Data collection and refinement statistics
Wild:MgATP
Wild:DLSA
Wild:AMP
S286N:DLSA
:Oxyluciferin
Data collection
Space group
P 212121
P 212121
P 212121
P 212121
57.8, 182.0, 53.6
57.6,181.3,52.0
57.6,181.4,52.7
57.4,181.1,52.2
 ()
90, 90, 90
90, 90, 90
90, 90, 90
90, 90, 90
Resolution (Å)
2.30
1.30
1.60
1.45
(2.38 – 2.30) *
(1.35 – 1.30)
(1.66 – 1.60)
(1.53 – 1.45)
Rsym or Rmerge
0.058 (0.150)
0.054 (0.209)
0.054 (0.210)
0.090 (0.283)
I/I
10.7 (4.0)
9.0 (2.6)
10.1 (3.0)
6.0 (2.6)
Completeness (%)
97.9 (91.1)
99.2 (95.8)
99.7 (97.4)
97.6 (95.0)
Redundancy
3.11 (2.55)
3.96 (3.36)
6.63 (3.58)
6.91 (7.47)
Resolution (Å)
2.30
1.30
1.60
1.45
No. reflections
25394
133611
73719
94838
Rwork/ Rfree
0.179 / 0.228
0.180 / 0.202
0.184 / 0.210
0.165 / 0.182
Protein
3976
4127
4111
4126
Ligand/ion
23
42
39
42
Water
283
794
626
843
Protein
23.5
10.0
15.1
9.9
Ligand/ion
13.6
5.4
7.6
5.0
Water
29.3
21.8
26.4
22.8
Bond lengths (Å)
0.011
0.009
0.011
0.010
Bond angles (º)
1.27
1.38
1.33
1.39
Cell dimensions
a, b, c (Å)
Refinement
No. atoms
B-factors
R.m.s deviations
*Highest resolution shell is shown in parenthesis.
Rmerge = |Ii-<Ii>| / Ii, where Ii = observed intensity, <Ii> = average intensity of multiple
measurements
14