452

Proc. Jpn. Acad., Ser. B 84 (2008)[Vol. 84,

Structure of commelinin, a blue complex pigmentfrom the blue ﬂowers of Commelina communis
By Masaaki SHIONO,1 Naohiro M ATSUGAKI2 and Kosaku T AKEDA3; y(Communicated by Shoji SHIBATA, M.J.A.)

Abstract: The X-ray crystal structure of natural commelinin is investigated. The results
demonstrate that commelinin is a tetranuclear (4 Mg2þ ) metal complex, in which two Mg2þ ionschelate to six anthocyanin molecules, while the other two Mg2þ ions bind to six ﬂavone
molecules, stabilizing the commelinin complex, a new type of supramolecular complex.
Keywords:
structurecommelinin, metal complex, blue pigment, anthocyanin, X-ray crystal

Introduction

Anthocyanin pigments responsible for ﬂower
colors, from red to blue,1),2) are generally isolated as
red oxonium salts using acid solvents. However,isolation of the native pigments without changing
colors and their characterization would be indispensable to clarify the mechanism of ﬂower color
variation. Commelinin, a blue pigment from theblue ﬂowers of Commelina communis L., was ﬁrst
isolated in 1957 using neutral solvents3) and wasshown to be a complex composed of a reddish purple
anthocyanin, a pale yellow ﬂavone and magnesium.4)–6) Bluing by chelation of a bivalent metal ion,
the Mg2þ ion, with anthocyanin was regarded assuspicious,7)–9) however, reconstruction of commelinin and metal-substituted commelinins using thecomponents demonstrated that a Mg2þ ion isessential.10)–12) X-ray analysis using reconstructed
Cd-commelinin11) indicated that commelinin was abinuclear (2 Mg2þ ) metal complex containing
anthocyanin and ﬂavone.13),14) However, the realfeatures of natural commelinin still remained obscure, because of diﬃculty of preparation of thecrystals needed for X-ray analysis.15) Recently wesucceeded in the preparation of single crystals of
1

Department of Physics, Graduate School of Science,
Kyushu University, Fukuoka, Japan.
2
Photon Factory, High Energy Accelerator Research
Organization, Ibaraki, Japan.
3Tokyo Gakugei University, Tokyo, Japan.
yCorrespondence should be addressed: K. Takeda,
Fukujima 1-9-17, Akishima, Tokyo 196-0031, Japan (e-mail:takedks@ss.iij4u.or.jp).

doi: 10.2183/pjab/84.452#2008 The Japan Academy

natural commelinin. Here we report its X-ray
crystal structure.
Material and methods

Natural commelinin was isolated from the fresh
petals of Commelina communis L. var. hortensis
Makino (Fig. 1a) principally in the same way asreported previously.4)–6) Press juice (3.1 l) from the
fresh petals (3.8 kg) was added with 6 vols. of
ethanol, whereby an amorphous blue pigment (ca5.5 g) was obtained. The pigment was further
puriﬁed by Sephadex (LH 20) column chromatography (2:5  35 cm) using water as an eluant andrepeated fractional precipitation from aqueous
ethanol solutions. The blue pigment was separated
from aqueous ethanol solutions in the form ofrectangular prisms. Recrystallization was repeatedmore than three times at 5  C. Finally, single
crystals available for X-ray analysis were separated.
Preparation of crystals of reconstructed and metal
substituted commelinins was carried out with thesame methods described previously,10),11) using
puriﬁed anthocyanin (AN) (malonylawobanin,16)delphinidin 3-O-(6-O-p-coumaroylglucoside)-5-O(6-O-malonylglucoside), Fig. 1b) and ﬂavone (FL)(ﬂavocommelin,17),18)
swertisin
40 -O-glucoside,
Fig. 1b).
All the crystals were sealed in glass capillaries
for diﬀraction measurements and data sets werecollected on BL-6A at the Photon Factory in KEK,
using an ADSC Quantum-4R CCD detector. The
data sets were processed with the HKL2000 package.19) All the crystals belong to space group P321

No. 10]Structure of commelinin

a

OH

b

3`

HO
O

O

O
5

HO

O
HO
HO

O

O
OH

4' O

B
3

HO
O

OH
OH

AN
HO
4' O

CH3O
HO
HO
HO

O
OH
O

O

3"

OH

O

OH
OH
OH

O
6

O
OH

OH

O

FL

Fig. 1. A blue ﬂower of Commelina communis and organic
components of commelinin. a, A blue ﬂower of Commelina
communis L. var. hortensis Makino. b, The components ofcommelinin, anthocyanin (AN): Malonylawobanin [delphinidin 3-O-(6-O-p-coumaroylglucoside)-5-O-(6-O-malonylglucoside)] and ﬂavone glycoside (FL): Flavocommelin [swertisin
40 -O-glucoside].

where an asymmetric unit contains one third ofa molecule (two ANs and two FLs). The initial
phase sets were obtained by an ab initio density
modiﬁcation method using program LODEM20),21)
and the structures were reﬁned with SHELXL97.22)
The ﬁnal R-factor for the natural commelinin
crystal was 0.1124 for 11786 reﬂections (> 4ðF Þ).
Figures were prepared using the program UCSF
Chimera.23)
Results and discussion

The crystal structure of natural commelinin
was determined at 0.95 Å resolution. The reﬁned
molecule has a three fold symmetry and four Mg2þions (Mg1Mg4) align along a three-fold axis
(Fig. 2a, b, Fig. 3a, b). The four metals arecoordinated to six AN molecules and six FL
molecules, forming an ellipsoidal shape with adimension of approximately 30 Å  30 Å  20 Å
(Fig. 2a, b). The distances among the four Mg2þions are Mg4-Mg1, 9.512 Å; Mg1-Mg2, 4.940 Å andMg2-Mg4, 6.781 Å respectively (Fig. 3a, b).

453

The inner two Mg2þ ions, Mg1 and Mg2 arecoordinated to the oxygen atoms of position 30 and
40 of the delphinidin nucleus of AN, in bonddistances of Mg1-O (30 ), 2.088 Å; Mg1-O (40 ),2.087 Å and Mg2-O (30 ) 2.091 Å; Mg2-O (40 )2.092 Å, respectively (Fig. 2b, Fig. 3a, 3c). On theother hand, Mg3 binds to the oxygen atom of
position 300 of glucose attached to position 40 of theswertisin nucleus of FL, in bond length of 2.115 Å
and three water molecules (W1) on the outersurface with the average distance of 2.031 Å
(Fig. 2b, Fig. 3b). Mg4 binds to the oxygen atomof position 300 (O3) of glucose attached to the 40
position of swertisin via a water molecule (W2) andto three water molecules (W3) (Fig. 2b, 3b). The
bond lengths of Mg4-H2 O(W2)-O(3) are 2.056 Å
and 2.715 Å, and that of Mg4-H2 O (W3) is 2.043 Å.
The temperature factors of the water molecules(W2 and W3) as well as Mg4 are small, and thus,
the contribution of the water molecules to the Mg4
and O(3) interaction is assumed to be signiﬁcant.
In the commelinin molecule, both AN and FL
are self-associated with each other as AN-AN and
FL-FL in pairs. The distances between the aromaticrings of each AN-AN and FL-FL are both approximately 3.3 Å, indicating hydrophobic interaction(Fig. 3a, b). The inner Mg1 and Mg2 ions are eachcoordinated to a diﬀerent AN fragment of theassociated AN-AN pair (Fig. 3a). Accordingly eachof the two Mg2þ ions binds with three ANs, one
from each of the three AN-AN pairs, forming athree-bladed propeller-like arrangement (Fig. 4a).
On the other hand, the outer two Mg2þ ions, Mg3
and Mg4, are each coordinated to a separate FL of
the associated FL-FL pair (Fig. 3b) and consequently two Mg2þ ions each bind to three FLs of
the associated FL-FL pairs in a three-bladed
propeller-like arrangement (Fig. 4b and 4c). In
addition, Mg3 and Mg4 coordinate to three watermolecules forming an octahedral geometry respectively (Fig. 4d and 4e).
The bond distances of the B-ring of AN in thecommelinin molecule are shown in Fig. 3c. These
data apparently indicate that the B-ring of AN is in
40 -keto-quinoidal form, and thus, AN chelates with
the Mg2þ ion in the form of a 40 -keto-quinoidal baseanion.
Commelinin was hitherto known as a binuclear
complex,13),14),24)–26) however, our results indicatethat natural commelinin is a tetra-metal nuclear

454

M. SHIONO, N. MATSUGAKI and K. TAKEDA[Vol. 84,ab

Fig. 2. Crystal structure of the commelinin molecule. Blue, anthocyanin (AN); yellow, ﬂavone glycoside (FL); green balls, Mg2þions; red balls, water molecules. a, Stereo view along the three-fold axis. b, A side view.

complex. To conﬁrm the total structure of commelinin, we reconstructed commelinin, Mg-commelinin and metal-substituted commelinin, Cd-commelinin, using puriﬁed AN and FL and metals in amanner similar to that described previously,10),11)
and further investigated by X-ray crystallography.
The results indicate that X-ray crystal structures of
the reconstructed Mg- and Cd-commelinins arebasically the same as that of natural commelinin.
Commelinin is known to be a stable blue
complex pigment even in acidic condition.4),5),12)
Natural commelinin showed a blue color andexhibited two characteristic absorption maxima inthe visible region, 589.1 nm and 644.1 nm, even in
10 mM acetic acid (pH 3.30) and also in 20 mM
potassium acetate buﬀer of pH 3.80. The spectra
were almost the same as those in water and acetate
buﬀer of pH 5.80. Copigmentation, the anthocyanin-copigment complex, is generally aﬀected by

pH.27) In fact, AN-FL (1:1) mixtures gave a purple
color at pH 5.80, having an absorption maximum at542.5 nm and a shoulder at 572.5 nm in the visible
region, while the color changed to purplish red(max 536.5 nm) in acetate buﬀer of pH 3.80 and tored (max 534.3 nm) in 20 mM acetic acid (pH 3.30).
We prepared the crystals of natural commelinin
from the solutions of 20 mM acetate buﬀer pH 3.80
and 10 mM acetic acid pH 3.30, by adding ethanol
respectively. X-ray structures of both samples werepractically the same as that described above andfour Mg2þ ions, Mg1Mg4, remained in the complex molecule. Furthermore, the occupation level ofthe outer two Mg2þ ions, Mg3 and Mg4, as well as
the inner two Mg2þ , Mg1 and Mg2, was notdecreased. The results indicate that the total
structure of the tetra-metal nuclear complex ofcommelinin remains constant even in acidic conditions.

No. 10]Structure of commelinin

a

b

455

c

Fig. 3. Metal binding sites in the commelinin molecule. Green balls, Mg2þ ions; red balls, water molecules. a, A side view of stacking
AN and AN. One AN binds to an inner Mg2þ ion, Mg1, while the other binds to another inner Mg2þ ion, Mg2. Blue, the front side
AN; cyan, back side AN. b, A side view of stacking FL and FL. One FL binds to an outer Mg2þ ion, Mg3, while the other binds toanother outer Mg2þ ion, Mg4, via water molecule, W2. Yellow, the front side FL; khaki, the back side FL. c, The bond distances(Å) of the B-rings of ANs in the commelinin molecule. Green balls, Mg2þ ions.

b

a

d

c

e

Fig. 4. Metal binding sites in the commelinin molecule. Green balls, Mg2þ ions; red balls, water molecules. a, A view along the threefold axis of an inner Mg2þ ion (Mg1 or Mg2) site. The bicolor frames, ANs. b, A view along the three fold axis of the outer Mg3
site. The bicolor frames, FLs. c, A view along the three fold axis of the outer Mg4 site. The bicolor frames, FLs. d, Mg3 bindingsite. Mg3 binds to the oxygen atom of position 300 of glucose attached to position 40 of the swertisin nucleus of FL and three watermolecules (W1) on the outer surface, forming octahedral geometry as shown by dotted line. e, Mg4 binding site. Mg4 binds to theoxygen atom of position 300 of glucose attached to the 40 position of swertisin via water molecule (W2) and to three watermolecules (W3), forming octahedral geometry as shown by dotted line.

456

M. SHIONO, N. MATSUGAKI and K. TAKEDAThe present work revealed the molecular
structure of natural commelinin by X-ray crystallography and demonstrated that the blue color isdeveloped by a homometallic tetra-nuclear complex
pigment comprising six AN molecules, six FL
molecules and four Mg2þ ions. The arrangement ofanthocyanins, ﬂavone glycosides and metal ions incommelinin is similar to that in protocyanin, a blue
complex pigment from Centaurea cyanus, which is aheterometallic tetra-nuclear (Fe3þ , Mg2þ , 2Ca2þ )complex pigment.28),29) In the commelinin molecule,the chelate formation of two Mg2þ ions with the 40 keto-quinoidal base of AN plays an important role
for the bluing and coordination of the other two
Mg2þ ions to FL has a crucial role for giving
stability to the complex molecule.

10)
11)
12)
13)
14)
15)
16)
17)
18)

Acknowledgements

We thank Dr. R. J. Grayer of the Jodrelllaboratory of Royal Botanic Garden Kew for
critical reading of the manuscript. K.T. acknowledges Dr. T. Iwashina of the laboratory of Tsukuba
Botanical Gardens for giving him an opportunity
to carry out part of this work at the laboratory.
The experiment at the Photon Factory was
adopted under the proposal No. 2002G320 and
No. 2006G19.
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(Received Oct. 29, 2008; accepted Nov. 12, 2008)