Enzymatic cleavage of the C-glucosidic bond of puerarin by three proteins, Mn2+, and oxidized form of nicotinamide adenine dinucleotide

Article abstract of DOI:10.1248/bpb.b12-01011

We previously isolated the human intestinal bacterium, strain PUE, which can cleave the C-glucosidic bond of puerarin to yield its aglycone daidzein and glucose. In this study, we partially purified puerarin C-glucosidic bond cleaving enzyme from the cell

Full text of DOI:10.1248/bpb.b12-01011

April 2013
Regular Article
Biol. Pharm. Bull. 36(4) 635–640 (2013)
635
Enzymatic Cleavage of the C-Glucosidic Bond of Puerarin by
2+
Three Proteins, Mn , and Oxidized Form of Nicotinamide Adenine
Dinucleotide
a,b
b
b
,a
Kenichi Nakamura, Katsuko Komatsu, Masao Hattori, and Makoto Iwashima*
a
ꢀ
FacultyꢀofꢀPharmaceuticalꢀSciences,ꢀSuzukaꢀUniversityꢀofꢀMedicalꢀScience;ꢀ3500−3ꢀMinamitamagaki,ꢀSuzuka,ꢀMieꢀ
b
5
13−8670,ꢀJapan:ꢀandꢀ ꢀInstituteꢀofꢀNaturalꢀMedicine,ꢀUniversityꢀofꢀToyama;ꢀ2630ꢀSugitani,ꢀToyamaꢀ930–0194,ꢀJapan.
Received November 21, 2012; accepted January 7, 2013; advance publication released online January 17, 2013
We previously isolated the human intestinal bacterium, strain PUE, which can cleave the C-glucosidic
bond of puerarin to yield its aglycone daidzein and glucose. In this study, we partially purified puerarin
C-glucosidic bond cleaving enzyme from the cell-free extract of strain PUE and demonstrated that the reac-
2
+
tion was catalyzed by at least three proteins, Mn , and oxidized form of nicotinamide adenine dinucleotide
+
(
NAD ). We completely purified one of the proteins, called protein C, by chromatographic separation in
three steps. The molecular mass of protein C was approximately 40kDa and the amino acid sequence of its
N-terminal region shows high homology to those of two putative proteins which belong to Gfo/Idh/MocA
family oxidoreductase. Protein C catalyzed hydrogen-deuterium exchange reaction of puerarin to 2″-deuter-
ated puerarin in D O condition, which closely resembles those of glycoside hydrolase family 4 and 109.
2
Key words puerarin; C-glucoside; C–C bond cleavage; glycosidase; enzyme purification
Glycosidic bonds between an aglycone and a sugar are to its aglycone daidzein, equol and related compounds by the
generally classified into four groups including O-, N-, S-, and intestinal bacteria. The latter pathway suggested that the key
C-glycosidic bonds. Glycoside hydrolases that catalyze one of reaction determining the metabolic fate of puerarin was C-gly-
the O, N, S-glycosidic bond hydrolysis are categorized into cosidic bond cleavage catalyzed by the intestinal bacteria. To
about 130 families based on the similarities of amino acid our best knowledge, a dozen of Pueraria isoflavones secondary
sequences (Carbohydrate-Active enZYmes database at http:// metabolized from puerarin show various and significant bio-
www.cazy.org/). Most glycoside hydrolases do not require any activities, however, the enzyme(s) performed the C-glycosidic
cofactors, however the glycoside hydrolase family 4 and 109 bond cleavage in puerarin as the initial key reaction has not
cleave glycosidic bonds through redox and elimination steps been characterized, yet.
under the specific reaction conditions involving oxidized form
of nicotinamide adenine dinucleotide (NAD ) as cofactor.
We previously isolated the human intestinal bacterium,
+
1,2)
strain PUE, which can cleave the C-glucosidic bond of puera-
17,18)
Quite a unique characteristic of C-glycosidic bond in C-gly- rin to yield daidzein and glucose
(Fig. 1). In the course of
cosides is found in the acidic hydrolysis or glycosidase treat- our investigation for the mechanistic study of biotransforma-
ments because their anomeric carbon in the sugar is directly tion of C-glycosides, the purification and characterization
attached to the aglycone by C−C bonding. Despite of the of the novel C-glycosidic bond cleaving enzyme from strain
chemical stability of C-glycosidic bond, biotransformation of PUE were partially accomplished. In this paper we describes
C-glycosides to its aglycone by the human intestinal bacteria an elucidation of the new enzyme composed of at least three
3
–12)
were reported.
A few papers described the partial purifica- proteins, and an existence of the cofactors associated with this
13,14)
tion of C-glycosidic bond cleaving enzymes.
to the paper of Sanugul et al., one of the enzymes for C- sequence of N-terminal region of one purified protein.
glycosidic bond cleavage reaction of mangiferin (xanthone C-
glucoside) consisted of two proteins which required Mn and MATERIALS AND METHꢀDS
undetermined low molecular cofactor(s).
According cleaving reaction. Furthermore, we determined the amino acid
14)
2+
C-Glycosides are distributed in a lot of medicinal plants.
General A bacterium was incubated in anaerobic incu-
Among them, puerarin, isoflavone C-glucoside, is a major bator EAN-140 (Tabai Co., ꢀsaka, Japan). UV spectra were
flavonoid contained in the roots of Pueraria lobata Ohwi measured by a UV-2200 UV/VIS recording spectrophotometer
(
Leguminosae), which is a well-known herbal drug Puerariae (Shimadzu Co., Kyoto, Japan).
Radix, so called “Kakkon” in Japanese and has been used as a Chemicals and Materials Strain PUE was isolated from
15)
17)
diaphoretic, antifebrile and antispasmodic in Japan and East human feces as previously described. Puerarin was isolated
Asia. from the roots of Pueraria lobata (willd.) Ohwi. General an-
After oral administration of puerarin in the case of rats, aerobic medium (GAM) broth was purchased from Nissui Sei-
16)
+
two metabolic pathways were reported by Prasain et al.
yaku Co. (Tokyo, Japan). NAD was purchased from ꢀriental
One showed that puerarin was first transferred to the portal Yeast Co. (Tokyo, Japan). Manganese(II) chloride tetrahydrate
circulation by the sodium-dependent glucose transporter, and was purchased from Wako Pure Chemical Industries, Ltd.
second entered itself into the bloodstream as an unmetabo- (ꢀsaka, Japan).
lized form. Another depicted that puerarin was metabolized
Preparation of Cell-Free Extract Strain PUE was cul-
tured under anaerobic conditions at 37°C for 14h in 1L of
GAM broth containing 0.3mm puerarin as enzyme inducer.
The authors declare no conflict of interest.
*
To whom correspondence should be addressed. e-mail: iwashima@suzuka-u.ac.jp
© 2013 The Pharmaceutical Society of Japan

6
36
Vol. 36, No. 4
Fig. 1. Cleavage of the C-Glucosidic Bond of Puerarin by Strain PUE
The bacterial cells were collected by centrifugation and sus- in a final volume of 100µL. It was incubated at 37°C for
pended in 50mL of 50mm potassium phosphate buffer (pH several hours, and extracted twice with 200µL of 1-butanol
7
.4). Cells were disrupted by sonication on ice and centrifuged (saturated with water and acidified with 0.1% acetic acid).
at 10000×g for 60min at 4°C to obtain a supernatant as cell- The extract was evaporated to dryness, dissolved in 50%
free extract. MeꢀH (100µL), and the amount of daidzein (metabolite) was
Purification of Puerarin C-Glucosidic Bond Cleaving measured by HPLC. HPLC conditions were as follows: col-
Enzyme (Proteins A, B, and C) All purification proce- umn, CꢀSMꢀSIL 5C -MS-II (Nacalai Tesque, Kyoto, Japan,
18
dures were carried out at 0–4°C. All column chromatography 4.6×150mm); flow rate, 1mL/min; detection, 256nm; mobile
steps for enzyme purification were performed using ÄKTA phase, 0.1% trifluoroacetic acid in 50% MeOH (isocratic); in-
purifier HPLC system (GE Healthcare, Buckinghamshire, jection volume, 20µL.
U.K.). Prepared cell-free extract was adjusted to 25% satura-
Determination of Cofactors The reaction mixture con-
tion of (NH ) Sꢀ followed by centrifugation at 10000×g sisted of partially purified proteins A (61µg), B (69µg), and
4
2
4
2+
for 10min. The supernatant was applied to a HiPrep Butyl C (21µg), puerarin (0.5mm), in the presence of both Mn
FF 16/10 column (GE Healthcare) at a flow rate of 5mL/min. (1 mm) and cofactor (NAD , reduced nicotinamide adenine
Bound proteins were eluted with a linear gradient of 1–0m dinucleotide (NADH), NAD phosphate (NADP ) or reduced
+
+
(
NH ) Sꢀ in 100mm potassium phosphate buffer (pH 7.3). NAD phosphate (NADPH), 1mm, respectively) or in the ab-
4 2 4
Two protein fractions (Fr. I and Fr. II) essential for the activity sence of either one, in a final volume of 100µL. After two
were pooled and further enzyme purification were performed hours incubation at 37°C, the enzyme activity was measured
as follows.
as described above.
Partial Purification of Proteins A and B from Fr. I: Fraction
Effects of Three Protein Fractions on Enzyme Activity
I was concentrated and buffer exchanged into 50mm potas- The reaction mixture consisted of a couple of partially puri-
sium phosphate buffer (pH 7.4) using Amicon Ultra-15 cen- fied proteins (A: 61µg, B: 69µg, C: 21µg), puerarin (0.5mm),
2+
+
trifugal filter devices (10K MWCO, Millipore). It was applied Mn (1 mm) and NAD (1 mm) in a final volume of 100µL.
to a Mono Q 4.6/100 PE column (GE Healthcare) at a flow rate After two hours incubation at 37°C, the enzyme activity was
of 1mL/min. Bound proteins were eluted with a linear gradi- measured as described above.
ent of 100–400mm NaCl in 50mm potassium phosphate buffer
Protein Measurement The protein content was deter-
(
pH 7.4). Two protein fractions (proteins A and B) essential mined by the method of Bradford, using Protein Quantifica-
for the activity were pooled.
tion Kit-Rapid (Dojindo Molecular Technologies, Inc., Kuma-
Complete Purification of Protein C from Fr. II: Fraction II moto, Japan).
was concentrated and buffer exchanged into 10mm potassium
Determination of Molecular Weight The molecular
phosphate buffer (pH 7.4) using Amicon Ultra-15 centrifugal weight of protein C was analyzed by sodium dodecyl sulfate-
filter devices. It was applied to a Hydroxyapatite-MP column polyacrylamide gel electrophoresis (SDS-PAGE) using c-Pagel
(
1
Kanto Chemical, Tokyo, Japan, 8×100mm) at a flow rate of 12.5% gel (ATTꢀ, Tokyo, Japan). Protein Molecular Weight
mL/min. Bound proteins were eluted with a linear gradient Marker (broad) (TaKaRa Bio Inc., Shiga, Japan) was used as
of 10–300mm potassium phosphate buffer (pH 7.4). The active standard. The protein bands were stained with Coomassie bril-
fractions were pooled and concentrated. It was applied to a liant blue R250.
Mono Q 4.6/100 PE column at a flow rate of 1mL/min. Bound
Determination of Amino Acid Sequence in N-Terminal
proteins were eluted with a linear gradient of 0–400mm NaCl Region After the solution of purified protein C was desalted,
in 50mm potassium phosphate buffer (pH 7.4). The active it was spotted onto a polybrene-coated glass fiber disk. The
fractions were pooled and used as a purified protein C in the amino acid sequence of its N-terminal region was determined
following experiments.
by the Edman degradation method using the protein sequencer
Measurement of Enzyme Activity Three protein frac- PPSQ-21 (Shimadzu, Kyoto, Japan). Homology searches were
2+
+
tions (proteins A, B and C), Mn and NAD were essential performed using the BLASTp search of the NCBI database.
for puerarin C-glucosidic bond cleaving reaction in our re- Hydrogen-Deuterium Exchange Reaction To the D ꢀ
sult. In the case of determining the active fraction of protein (870 µL) was added purified protein C (43µg), puerarin
2
2+
+
C, beforehand prepared proteins A and B were added to the (0.5 mm), Mn (1 mm) and NAD (1 mm) in a final volume of
reaction mixture. The reaction mixture consisted of enzyme 1mL (87% D ꢀ condition). After the reaction mixture was
2
+
solution, puerarin (0.5mm), MnCl (1 mm) and NAD (1 mm) incubated at 37°C for 20h, it was extracted three times with
2

April 2013
637
500µL of 1-butanol (saturated with water and acidified with Varian 400-MR (Varian Inc., CA, U.S.A.).
0
.1% acetic acid). The extract was evaporated to dryness, dis-
1
solved in CD ꢀD and H-NMR of the residue was taken on RESULTS
3
Effects of Cofactors on Enzymatic Activity The cell-
free extract of strain PUE showed weak puerarin C-glucosidic
2+
bond cleaving activity, while by addition of Mn to the reac-
tion mixture the activity was significantly increased. From the
2+
follow-up study, we found that the presence of Mn together
with some low molecular cofactor(s) furnished more acceler-
ated reaction. In order to identify the cofactor(s), we tried to
use combined cocktail of the partially purified enzyme frac-
tions for C–C bond cleaving reaction (Fig. 2). The best condi-
2
+
+
tion was found in the presence of both Mn and NAD , but in
2+
+
the absence of either Mn or NAD , the cleavage underwent
fairly. The effect of NADH, NADP , and NADPH were also
+
examined in the same conditions, however, these cofactors
showed a slight effect on the cleaving activity.
Partial Purification of Puerarin C-Glucosidic Bond
Fig. 2. Effects of Cofactors on Puerarin C-Glucosidic Bond Cleaving Cleaving Enzyme (Proteins A, B, and C) We purified
Activity of Partially Purified Enzyme
puerarin C-glucosidic bond cleaving enzyme from the cell-
free extract of strain PUE by using column chromatography
in several steps (Fig. 3). First step column chromatography
was performed using a HiPrep Butyl FF 16/10 column (Fig.
The reaction mixture consisted of partially purified proteins A (61 µg), B (69µg),
2+
and C (21µg), puerarin (0.5mm), in the presence of both Mn (1 mm) and cofactor
+
+
(
NAD , NADH, NADP or NADPH, 1mm, respectively) or in the absence of either
one, in a final volume of 100µL. After 2h incubation at 37°C, the enzyme activity
was measured.
Fig. 3. Purification of Puerarin C-Glucosidic Bond Cleaving Enzyme
The arrow represents the active protein fractions (proteins A, B, and C). (a) HiPrep Butyl FF 16/10 column chromatography of cell-free extract, (b) Mono Q 4.6/100
PE column chromatography of Fr. I, (c) Hydroxyapatite-MP column chromatography of Fr. II, (d) Mono Q 4.6/100 PE column chromatography of the active fraction from
hydroxyapatite-MP.

6
38
Vol. 36, No. 4
3a), to yield in disappearance of the bond cleaving activity in for C-glucosidic bond cleaving reaction. We used partially
any of one fraction alone. However, the activity was restored purified proteins A (61µg), B (69µg), and C (21µg). If the
2+
in the mixed condition of two fractions (Fr. I and Fr. II), Mn
three proteins were combined with cofactors, puerarin C-
and NAD , indicating that at least two protein fractions were glucosidic bond cleaving activity was 12.5nmol/h. The ab-
essential for the bond cleaving reaction.
sence of any proteins resulted in remarkable decrease of the
+
Further enzyme purification of Fr. I was conducted using activity, which suggested that all of the three proteins were
a Mono Q 4.6/100 PE column (Fig. 3b). We found that the exactly essential for the bond cleaving reaction. We had tried
essential protein contained in Fr. I was not of single protein to identify any reaction intermediate such as daidzin (daidzein
and they were separated again to two protein fractions, which
were called proteins A and B, respectively. The other essential
protein contained in Fr. II was called protein C. At this stage
of enzyme purification, the proteins A, B, and C were par-
tially purified proteins.
Figure 4 shows the requirement of three protein fractions
Fig. 4. Requirement of Three Protein Fractions for C-Glucosidic Bond
Cleaving Reaction
Fig. 5. SDS-PAGE of the Protein C ꢀbtained by Various Steps of Col-
umn Chromatography
The reaction mixture consisted of a couple of partially purified proteins (A:
1 µg, B: 69µg, C: 21µg), puerarin (0.5mm), Mn (1 mm), and NAD (1 mm) in
a final volume of 100µL. After 2h incubation at 37°C, the enzyme activity was
measured.
Lane M, marker proteins; lane 1, cell-free extract; lane 2, the active fraction (Fr.
II) obtained from HiPrep Butyl FF 16/10 column chromatography; lane 3, the ac-
tive fraction from Hydroxyapatite-MP column chromatography; lane 4, the active
fraction from Mono Q 4.6/100 PE column chromatography.
2+
+
6
Fig. 6. Hydrogen-Deuterium Exchange Reaction Catalyzed by Protein C
1
1
2+
+
(A) H-NMR spectrum of puerarin standard, (B) H-NMR spectrum of puerarin after incubation with protein C, Mn , and NAD in 87% D ꢀ condition.
2

April 2013
639
7
-O-β-d-glucopyranoside) by HPLC analysis after incubation acid sequence of its N-terminal region (Fig. 5). Hydrogen-
of puerarin with the partially purified protein(s), but unfortu- deuterium exchange reaction was observed at the C-2ꢀ of pu-
nately we could not detect it yet. erarin when it was incubated with protein C in D ꢀ condition
Complete Purification and Amino Acid Sequence of (Fig. 6). The results is closely resembled those of glycoside
2
1,2)
N-Terminal Region of Protein C We purified protein C hydrolase family 4 and 109. These glycoside hydrolases in-
from the cell-free extract of strain PUE by chromatographic volve in redox reaction of carbohydrate at C-3 using NAD as
+
separation in three steps, such as a Butyl FF column (Fig. 3a), cofactor. After the oxidation of hydroxyl at C-3, hydrogen at
Hydroxyapatite-MP column (Fig. 3c) and Mono Q column C-2 was exchanged to deuterium from D ꢀ because the acid-
2
(
Fig. 3d). The active fraction obtained from the purification ity of H-2 is increased by an adjacent carbonyl group. Finally,
by the Mono Q column showed single band of approximately the reaction was completed by reduction of ketone at C-3 to
0kDa on SDS-PAGE (Fig. 5) and the amount of obtained hydroxyl using the hydride generated from NADH. The en-
4
protein C was 430µg.
zymatic function of protein C showed some similarities to the
glycoside hydrolase family 4 and 109, reasonably supporting
The N-terminal amino acid sequence of protein
C
18)
was determined by the Edman degradation method as the fact that puerarin is hydrolyzed to daidzein and glucose.
SKLKIGIIGXGGIANQKHFPALKNNADLNE. BLASTp
Puerarin C-glucosidic bond cleavage reaction still remained
search showed that the amino acid sequence of N-terminal re- some unknown reaction mechanism such as unclear roles
A
2+
gion showed high homology to those of two putative proteins; of three proteins, together with the interaction of Mn and
+
9
3% identity with oxidoreductase, Gfo/Idh/MocA family from NAD . Protein C alone could not cleave the C-glucosidic bond
Clostridium sp. D5 (ZP_08128353.1) and 90% identity with without proteins A and B. Further studies will be needed to
hypothetical protein HMPREF0988_02145 from Lachnospi- conclude the mechanism of puerarin C-glucosidic bond cleav-
raceae bacterium 1_4_56FAA (ZP_08616560.1). Both putative age reaction, and to complete the purification and character-
proteins belong to the Gfo/Idh/MocA family oxidoreductases, ization of proteins A and B.
19)
in which represented glucose-fructose oxidoreductase, myo-
2
0)
2)
inositol 2-dehydrogenase, glycoside hydrolase family 109,
and so on.
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