2418
M. SAKAI et al.
Cosmosil PAQ column (4:6 ꢃ 150 mm, Nacalai Tesque)
were used. The solvents were A: 0.1 M NaOAc contain-
ing 5.6 mM triethylamine (pH 5.8), and B: MeCN. The
column was developed by increasing the latter from
PAld, acetophenone, 2-phenylpropionaldehyde, 3-phe-
nylpropionaldehyde, salicylaldehyde, 4-hydroxybenzal-
dehyde, citral, hexylaldehyde, benzaldehyde, trans-cin-
namaldehyde, (R)-(+)-citronellal, (S)-(ꢂ)-citronellal, 2-
butanone, methyl butylketone and methyl pentylketone.
3
0% to 35% in 30 min, then to 90% in 35 min at a flow
ꢁ
rate of 0.8 ml/min at 40 C. The excitation and emission
wavelengths were 315 and 270 nm, respectively. To
analyze the effects of the coenzymes, PLP and/or
NADPH were added in various concentrations, as shown
in the legend to figures. Iproniazid (Sigma-Aldrich) was
used as an inhibitor of MAO.
This work was supported in part by grant-aid to N.W.
for scientific research (B)(2) from the Ministry of
Education, Culture, Sports, Science and Technology
(MEXT) of Japan, and grant-aid to N.W. and M.H. from
Cooperation of Innovative Technology and Advanced
Research in Evolutional Area (city area) in Central
Shizuoka, MEXT of Japan.
We also express thanks to Prof. Yamaki of Nagoya
University in Japan, for his informative discussions on
the purification of the enzymes.
Detection of H2O2 in the enzymatic reaction mixture.
The partially purified rose petal AADC fractions were
replaced with a 0.01 M potassium phosphate buffer
(
method just mentioned. The production of H O in the
pH 7.0). The enzyme reaction was conducted by the
2
2
reaction mixture was assayed with FOX reagent as
2
2)
described by Wolff.
References
Expression of rose petal AADC in Escherichia coli.
The cDNA for rose petal AADC was cloned into the
pET28a expression vector (Novagen) to generate a
construct coding for a protein with an N-terminal His
tag. The rose petal AADC cDNA was amplified from a
rose EST clone2 and cloned into pET28a to code for a
protein with a C-terminal His tag. The construct was
verified by DNA sequencing. Recombinant AADC
expressed in E. coli was purified using a His-Tag
column (1 ꢃ 2 ml, GE Healthcare). The protein concen-
tration was determined by the Bradford method, using
bovine serum albumin as a standard.
1
2
)
)
Clark, G. S., Phenetyl alcohol. Perf. Flav., 15, 37–44
1990).
(
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beta-phenylethanol in rose petals. Biokhimiia, 43, 2038–
0)
3)
4)
5)
2
042 (1978).
Substrate specificity of AADC and PAR. In assays to
determine the substrate specificity of AADC, the
enzyme activity was assayed by measuring the NH3
production in the reaction mixture after 60 min, as
already described. The substrates used were L- and D-
Phe, L-tyrosine, L-arginine, L-tryptophane, and L-valine.
PAR activity was assayed by estimating the amount of
Albertazzi, E., Cardillo, R., Servi, S., and Zucchi, G.,
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)
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Watanabe, S., Hayashi, K., Yagi, K., Asai, T.,
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2
þ
incorporation of [ H8] L-phenylalanine into 2-phenyl-
NAD in the reaction mixture after 30 min. The reaction
mixture contained 40 ml of an enzyme solution with
ethanol and its ꢀ-D-glucopyranoside during the flower
opening of Rosa ‘Hoh-Jun’ and Rosa damascena Mill.
Biosci. Biotechnol. Biochem., 66, 943–947 (2002).
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2-phenylethanol from its ꢀ-D-glucopyranoside and the
2
0 ml of substrate (10 mM each), 20 ml of NADH (10 mM)
and 120 ml of 0.1 M potassium phosphate buffer (pH 7.0).
At the end of the reaction, 300 ml of MeCN and 50 ml of
hippuric acid (25 mg in 100 ml of the phosphate buffer)
were added. After filtration through a membrane
7
2
(
0.45 mm), 10 ml of the sample solution was assayed by
biogenesis of these compounds from [ H8] L-phenyl-
alanine in rose flowers. Tetrahedron, 60, 7005–7013
HPLC under the following conditions: A Waters 2695
Separations Module equipped with a Waters 2996
Photodiode Array Detector and a Mightysil RP-18 GP
Aqua column (4:6 ꢃ 150 mm, Kanto Chemical) were
used. The solvents were A: 50 mM phosphate buffer
(
2004).
8
)
)
Wittstock, U., and Halkier, B. A., Cytochrome P450
CYP79A2 from Arabidopsis thaliana L. catalyzes the
conversion of L-phenylalanine to phenylacetaldoxime in
the biosynthesis of benzylglucosinolate. J. Biol. Chem.,
(
increasing the latter from 2% to 15% in 5 min, and then
pH 6.0) and B: MeCN. The column was developed by
2
75, 14659–14666 (2000).
9
Tieman, D., Taylor, M., Schauer, N., Fernie, A. R.,
Hanson, A. D., and Klee, H. J., Tomato aromatic amino
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volatiles 2-phenylethanol and 2-phenylacetaldehyde.
ꢁ
to 60% in 9 min at a flow rate of 1 ml/min at 35 C. The
þ
wavelengths were 258 and 230 nm for NAD and
hipurric acid, respectively. The substrates used were