Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.)

Article abstract of DOI:10.1016/j.phytochem.2004.08.006

A cDNA encoding adenylate isopentenyltransferase was cloned and sequenced from Humulus lupulus L. The corresponding recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from DMAPP to the N⁶ amino group of AMP, ADP and ATP, respectively. Site-directed mutagenesis of a conserved Asp62 resulted in complete loss of enzyme activity. A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop (Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38-51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N⁶ amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity.

Full text of DOI:10.1016/j.phytochem.2004.08.006

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2439–2446
www.elsevier.com/locate/phytochem
Molecular cloning, expression, and characterization of adenylate
q
isopentenyltransferase from hop (Humulus lupulus L.)
a
b
a
a
Yuichi Sakano , Yukio Okada , Akiko Matsunaga , Takaharu Suwama ,
b
Takafumi Kaneko , Kazutoshi Ito , Hiroshi Noguchi , Ikuro Abe
b
a
a,
*
a
School of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
b
Plant Bioengineering Research Laboratories, Sapporo Breweries Ltd., 37-1 Kizaki, Nitta, Gunma 370-0393, Japan
Received 24 April 2004; received in revised form 2 July 2004
Abstract
A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop (Humulus lupulus L.)
by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1,
AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-bp open reading frame encoding
a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of
introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demon-
strated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced
amino acid sequence shares 38–51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli
catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N6 amino group of adenosine monophos-
phate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guan-
osine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP),
were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP
were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative
ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a
conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Humulus lupulus; Cannabidaceae; Adenylate isopentenyltransferase; Prenylation; Cytokinin
1. Introduction
group of phytohormones that play a critical role in
plant growth and development (Mok and Mok,
2001). Most natural cytokinins are derivatives of N6-
prenylated adenine, and at least isopentenyladenine
and trans-zeatin are regarded as active forms in plants.
Only recently, the Arabidopsis genome project revealed
the sequences of AIPTs in Arabidopsis thaliana. The
plant contains nine isozymes (AtIPT1, AtIPT2,
AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, AtIPT8
and AtIPT9) sharing sequence similarity with tRNA
isopentenyltransferases (IPTs) from bacteria, prokaryotes
Adenylate isopentenyltransferase (AIPT) (EC
2.5.1.27) is a pivotal enzyme in the biosynthesis of cyto-
kinins (Fig. 1) (Takei et al., 2001; Kakimoto, 2001), a
q
The nucleotide sequence reported in this paper is available in the
DDBJ/EMBL/GenBankTM data bases under the Accession No.
AY533024.
*
Corresponding author. Tel./fax: +81 54 264 5662.
E-mail address: abei@ys7.u-shizuoka-ken.ac.jp (I. Abe).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.08.006

2440
Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
Fig. 1. Prenyl transfer reaction of adenosine phosphate (AMP, ADP or ATP) to isopentenyl adenosine phosphate by AIPT in H. lupulus.
and eukaryotes (Takei et al., 2001; Kakimoto, 2001).
So far, AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6,
AtIPT7 and AtIPT8 have been shown to be involved
in the de novo biosynthesis of cytokinins. Thus, rec-
ombinant enzymes heterologously expressed in Escheri-
chia coli catalyzed transfer of an isopentenyl group
from dimethylallyldiphosphate (DMAPP) to the N6
amino group of adenosine phosphates (Takei et al.,
2001; Kakimoto, 2001; Sakakibara and Takei, 2002;
Sun et al., 2003). Although it has been pointed out that
AIPT enzymes contain the putative ATP/GTP binding
motif, structural details of the enzyme reaction still re-
main to be elucidated.
The presence of multiple AIPTs in A. thaliana sug-
gested the differentiation of the physiological function
of each isoform. Since some plant enzymes exhibit fairly
broad substrate specificities, it would be possible that
some of the AIPT isozymes are involved in the biosyn-
thesis of secondary metabolites such as prenylated chal-
cones and prenylated phloroglucinols. Although these
metabolites are considered to derive from prenylation
of precursor compounds, the responsible enzymes in-
volved in the isopentenyl transfer reactions have not
been identified yet.
The cones of the hop plant (Humulus lupulus L.,
Cannabinaceae) have been used for centuries in the
beer-brewing process as a source of lupulin compo-
nents. The plant produces significant amount of bitter
acids, highly prenylated phloroglucinols (e.g., humu-
lone and lupulone), as well as prenylated chalcones
(e.g. xantohumol), for which anti-microbial, anti-fungal
and anti-cancer activities have been reported (Stevens
et al., 2000; Zuurbier et al., 1998; Paniego et al.,
1999; Okada and Ito, 2001; Okada et al., 2001). To study
the biosynthesis of cytokinins as well as to shed light on
the biogenesis of the prenylated secondary metabolites
in H. lupulus, we carried out PCR cloning of AIPT
cDNA using oligonucleotide primers based on the se-
quences of A. thaliana AIPTs. This is the first report
of molecular cloning and characterization of a plant
AIPT except those for A. thaliana. Functional analyses
of a recombinant enzyme including enzyme kinetics,
substrate specificity and site-directed mutagenesis, are
also presented.
2. Results and discussion
2.1. Molecular cloning of cDNA encoding AIPT
A cDNA encoding AIPT was cloned and sequenced
from cones of H. lupulus by RT-PCR using inosine-con-
taining degenerate oligonucleotide primers based on the
highly conserved sequences of A. thaliana AIPT iso-
zymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6,
AtIPT7 and AtIPT8) (Takei et al., 2001; Kakimoto,
2001) (Fig. 2). The terminal sequences of cDNA were
obtained by 30-RACE and 50-inverse PCR methods. A
1642-bp full-length cDNA contained a 508-bp 50 non-
coding region, a 990-bp open reading frame encoding
a molecular mass of 36,603 Da protein with 329 amino
acids, and a 144-bp of 30 non-coding region. Further,
genomic DNA was also sequenced by the 50-inverse
PCR and PCR using genomic DNA as template. Inter-
estingly, the gDNA sequence revealed absence of introns
in the frame. On Southern blot analysis, a single band
was detected (Fig. 3), suggesting presence of a single
copy AIPT gene in H. lupulus, which is in sharp contrast
with the case of A. thaliana that contains the nine AIPT
genes. Moreover, RT-PCR analyses revealed that the
gene was equally expressed in almost all tissues in
H. lupulus including roots, stems, leaves and cones
(Fig. 4), while it has been reported that A. thaliana
AtIPT8 transcript was only detected in root tissues
(Sun et al., 2003).
The deduced amino acid sequence of H. lupulus
AIPT showed 51%, 39%, 47%, 39%, 46%, 38% and
49% identity to those of AIPT isozymes from A. thali-
ana, AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6,
AtIPT7 and AtIPT8, but exhibited only 34% and 17%
identity to those of A. thaliana tRNA IPT isozymes,
AtIPT2 and AtIPT9, respectively (Fig. 2). H. lupulus
AIPT contains GATGTGKS (amino acids number
37–44) sequence known as the ATP/GTP binding motif
which is universally observed in ATP-consuming en-
zymes including ATP-binding cassette transporters (Ta-
kei et al., 2001; Kakimoto, 2001). On the other hand,
the putative zinc finger-like motif designated as C–X2–
C–X{12,18}–H–X5–H (where X denotes any amino
acid residue, and X{m, n} m to n amino acid residues

Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
2441
Fig. 2. Comparison of deduced amino acid sequences of H. lupulus AIPT and A. thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5,
AtIPT6, AtIPT7 and AtIPT8).
in number) which is conserved in A. thaliana AtIPT2
and eukaryotic tRNA IPTs is not present in H. lupulus
AIPT as in the case of A. thaliana AIPT isozymes (Ta-
kei et al., 2001; Kakimoto, 2001). Furthermore, like
other IPTs, H. lupulus AIPT shares, right downstream
of the ATP/GTP binding motif, a consensus pattern
of X5-(V/L/I)-X7-(V/L/I)(V/L/I)-X2-D-X2-Q-X {57,60}-
(V/L/I)(V/L/I)-X-GG-(S/T) (where X denotes any ami-
no acid residue, and X{m, n} m to n amino acid residues
in number) (Takei et al., 2001; Kakimoto, 2001). Within
the ATP/GTP binding motif and the consensus region,
H. lupulus AIPT exhibited 64%, 55%, 66%, 56%, 62%,
54% and 62% identity to those of AIPT isozymes in
Fig. 3. Southern blot analysis of H. lupulus AIPT. Genomic DNA was
digested with: lane 1, Bam HI; lane 2, Bgl II; lane 3, Dra I; lane 4, Eco
A. thaliana, AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6,
AtIPT7 and AtIPT8 and these scores paralleled well
those of the overall sequence. However, the NXDDX
or DDXXD putative diphosphate binding motif seen
in many prenyltransferases was not found in H. lupulus
AIPT as well as other IPTs, while Asp62, which was lo-
cated 18-amino-acid downstream from the ATP/GTP
binding motif, was strictly conserved among all of
known IPT enzymes, suggesting possible involvement
in phosphate binding via salt bridge of magnesium diva-
lent cation. Indeed, our site-directed mutagenesis exper-
iment demonstrated that replacement of Asp62 with
Ala resulted in complete loss of enzyme activity.
T22I; lane 5, Vsp I. The positions and sizes (indicated as kilobase-
pairs) of molecular size marker (kDNA; digested with Hind III) were
shown on the left.
In the phylogenetic tree (Fig. 5) (Thompson et al.,
1994), the AIPT from H. lupulus (Cannabinaceae) forms
a cluster with A. thaliana AIPTs (AtIPT1, AtIPT3,
AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8) and Lo-
tus japonicus AW720363 (EST clone), but a separate
Fig. 4. RT-PCR analysis of expression pattern of H. lupulus AIPT:
lane 1, root; lane 2, stem; lane 3, leaf; lane 4, cone. The positions and
sizes (indicated as kilobase-pairs) of molecular size marker (kDNA;
digested with Hind III), and the position of amplified ca. 0.9-kb DNA
bands (indicated by arrow) were shown on the left.

2442
Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
Fig. 5. Phylogenetic tree of the IPT enzymes. Multiple sequence alignment performed by CLUSTAL W (1.8). The three EST clones (L. japonicus
AW720363, M. truncatula AW691664 and S. tuberosum BE921755) are indicated by asterisk.
cluster with other IPT enzymes including A. thaliana
tRNA IPT (AtIPT2 and AtIPT9) and IPTs from
plant-pathogenic bacteria. Further, a putative AIPT
clone obtained from Morus alba (Moracerae) in our lab-
oratories (Abe, I. and co-workers, unpublished data) is
also included in the tree. The H. lupulus and M. alba
AIPTs appear to be evolutionary more related to
AtIPT1, AtIPT4, AtIPT6 and AtIPT8 than to AtIPT3,
AtIPT5 and AtIPT7 isoforms.
M15[pREP4] with an additional hexahistidine-tag at the
N-terminal. Purification by Ni-chelate affinity column
chromatography afforded ca. 1 mg of homogeneous rec-
ombinant AIPT from 1 g of E. coli cell pellet. The puri-
fied enzyme was first incubated with AMP and DMAPP
as substrates under the assay condition as previously re-
ported for A. thaliana AIPT isozymes (Takei et al., 2001;
Kakimoto, 2001). The reaction products were directly
analyzed by reverse phase HPLC, which clearly demon-
strated the formation of isopentenylated product in
comparison with the control reaction using boiled
enzyme. Thus, H. lupulus AIPT catalyzed the transfer
of an isopentenyl group from DMAPP to the N6
amino group of AMP (Fig. 1). The enzyme activity of
2.2. Functional expression of AIPT
The 990-bp H. lupulus AIPT cDNA was cloned into
pQE-80L (Qiagen) and functionally expressed in E. coli

Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
2443
Table 1
Steady-state kinetic parameters for enzyme reactionsa
to elucidate the structural details of the isopentenyl
transfer reaction from DMAPP to the N6 amino group
of adenosine phosphate, and to manipulate the cytoki-
nin biosynthesis in the plant, further characterization
of the enzyme is now in progress in our laboratories.
Substrate
kcat (minꢀ1
)
KM (lM)
kcat/KM (sꢀ1 Mꢀ1
)
AMP
ADP
ATP
DMAPP
a
0.497 0.009
1.43 0.06
1.31 0.09
1.63 0.10
759 40
10.90
1238
1340
1397
19.3 2.8
16.2 3.2
19.5 3.2
Michaelis–Menten plots of data were employed to derive the KM
and kcat values (average of duplicates standard deviation) using
EnzFitter software (BIOSOFT).
4. Experimental
4.1. Chemicals
Dimethylallyldiphosphate, adenosine triphosphate,
adenosine diphosphate, adenosine monophosphate,
H. lupulus AIPT showed dependence on magnesium
divalent cation and a broad pH optimum between 6
and 9, either in Tris–HCl or potassium–phosphate buf-
fer systems. On the other hand, other ribonucleotides;
guanosine monophosphate (GMP), inosine monophos-
phate (IMP), cytosine monophosphate (CMP) and uri-
dine monophosphate (UMP), were not accepted as a
substrate, suggesting that at least the purine core struc-
ture and the N6 amino functional group is essential for
the isopentenyl transfer activity.
guanosine
monophosphate,
inosine
monophos-
phate, cytosine monophosphate, and uridine monophos-
phate, were purchased from Sigma. [1-3H] DMAPP
(triammonium salt; 20 Ci/mmol) was from American
Radiolabeled Chemical, Inc.
4.2. Plant materials
It has been reported that some A. thaliana AtIPT en-
zymes such as AtIPT4 preferred adenosine diphosphate
(ADP) and adenosine triphosphate (ATP), rather than
adenosine monophosphate (AMP), as a prenyl acceptor
substrate (Takei et al., 2001; Kakimoto, 2001). Steady-
state kinetic analyses of H. lupulus AIPT revealed that
the isopentenylation of ADP and ATP were more effi-
cient than that of AMP as in the case of A. thaliana
AtIPT4, but contrast with Agrobacterium tumefaciens
TZS which only recognizes AMP as acceptor (Morris
et al., 1993). The observed KM and kcat parameters were
as follows (Table 1); 759 lM and 0.497 minꢀ1 for
AMP, 19.3 lM and 1.43 minꢀ1 for ADP, 16.2 lM
and 1.31 minꢀ1 for ATP, and 19.5 lM and 1.63 minꢀ1
for DMAPP, respectively. The KM values for ATP and
DMAPP in this enzyme were comparable to or a little
higher than those of AtIPT4. The kcat/KM values for
AMP, ADP, ATP and DMAPP were 10.90, 1238,
1340 and 1397 sꢀ1 Mꢀ1, respectively. The kcat/KM value
for ATP was thus 123-fold higher than that of AMP.
This suggested that cytokinins are also generated from
ATP and ADP in hop plant as in the case of
A. thaliana.
All plant tissues were prepared from H. lupulus L.
(9418R). Cones were harvested 15 days after flowering
from plants grown in the field of Sapporo Breweries,
Ltd., Japan. All plant materials were frozen in liquid
nitrogen and stored at ꢀ80 ꢁC.
4.3. PCR and DNA sequencing
The polymerase chain reaction (PCR) was carried out
with Ex-Taq DNA polymerase (Takara) using Robocy-
cler Gradient 40 (Stratagene). The nucleotide sequences
were determined from both strands by DNA sequencer
ABI 373A (Perkin–Elmer Applied Biosystems) using
Thermo SequenaseTM II Dye Terminator Cycle
Sequencing Kit (Amersham Biosciences).
4.4. Preparation of genomic DNA
Genomic DNA (gDNA) was prepared from the
leaves by a modified CTAB method (Murray and
Thompson, 1980). The 2% CTAB solution (2% cetyltri-
methylammonium bromide, 0.1 M Tris, 20 mM EDTA,
1.4 M NaCl, and 5% 2-mercaptoethanol, pH 9.5) was
added to the ground leaves, mixed, and incubated at
65 ꢁC for 30 min. The solution was extracted with
CHCl3 twice, and DNA was precipitated by adding
an equal volume of 2-propanol to the aqueous phase.
The precipitated DNA was dissolved in High Salt TE
(10 mM Tris, 1 mM EDTA, and 1 M NaCl, pH 8.0)
and treated with RNase at 65 ꢁC for 30 min. The
DNA was precipitated by adding an equal volume of
2-propanol to the solution and rinsed with 70% aque-
ous ethanol. The DNA was dried and dissolved in dis-
tilled water.
3. Conclusions
In summary, this is the first report of molecular clon-
ing of AIPT enzyme from H. lupulus, except those for A.
thaliana whose sequences were identified by the genome
project. Functional analyses of a recombinant enzyme
including enzyme kinetics, substrate specificity and
site-directed mutagenesis, provided important informa-
tion on the catalytic function of the enzyme. In order

2444
Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
4.5. Preparation of total RNA and cDNA
810-bp DNA fragment. On the other hand, the 50-end
sequence was finally obtained by the 50 inverse PCR
method. Thus, the genomic DNA prepared from leaves
was first digested with Psh BI (Takara), and circularized
using the DNA Ligation Kit (Takara) according to the
manufacturerÕs instruction, which was then used as a
template for the PCR reactions using four specific prim-
ers based on the obtained core sequence. The sequences
of the primers are as follows: 142A = 50-TC AAA CCG
GTC CAC CAA CAG-30, 170A = 50-AC CCA GAG
AAA GCA ACA GTC-30, 178S = 50-ACC GAT TAC
TTG GCG AAG GC-30, and 196S = 50-AG TTG
GCC GAG TTC TAT AGC-30. Nested PCR was car-
ried out with the primer sets of 170A and 178S, and then
with 142A and 196S, to amplify a 1,450-bp DNA frag-
ment. For the PCR, 30 cycles of reactions (94 ꢁC for
0.5 min, 55 ꢁC for 0.5 min and 72 ꢁC for 6 min) were per-
formed each time with a 7 min final extension.
The total RNA of the cone was prepared by the
modified CTAB procedure (Mukai and Yamamoto,
1995). The 2% CTAB solution (2% cetyltrimethylam-
monium bromide, 0.1 M Tris, 20 mM EDTA, 1.4 M
NaCl, and 5% 2-mercaptoethanol, pH 9.5) was added
to each ground tissue, mixed, and incubated at 65 ꢁC
for 30 min. The solution was extracted with CHCl3
twice, and RNA was precipitated by adding one-fourth
volume of 10 M LiCl to the aqueous phase. The precip-
itated RNA was dissolved in DNase buffer (100 mM
Na-Acetate, 5 mM MgCl2, pH 5.2) and treated with
DNase (RNase free) at 30 ꢁC for 60 min. After phe-
nol-CHCl3 extraction, the RNA was precipitated by
adding one-fourth volume of 10 M LiCl to the solution,
then rinsed with 70% aqueous ethanol. The RNA was
dried and dissolved in distilled water, then reverse tran-
scribed using SuperScriptTM (Invitrogen) and oligo dT
primer (RACE 32 = 50-GAC TCG AGT CGA CAT
CGA TTT TTT TTT TTT TT-30) according to manu-
facturerÕs protocol. The obtained cDNA mixture was
diluted with TE (10 mM Tris, 1 mM EDTA, pH 8.0)
and used as a template for the following PCR
amplification.
4.7. Southern blot analysis
Genomic DNA (10 lg) was digested with Bam HI,
Bgl II, Dra I, Eco T22I, and Vsp I separated on a 1%
agarose gel and blotted onto a nylon membrane. The
recognition sites of all of these restriction enzymes were
absent in the structural gene of H. lupulus AIPT.
Hybridization with H. lupulus AIPT cDNA was per-
formed using the DIG system (Roche Diagnostics)
according to the manufacturerÕs instructions. Hybridiza-
tion was done at 68 ꢁC overnight with the digoxinlabeled
probe in 5· saline sodium citrate (SSC), 50% forma-
mide, 0.02% SDS, 0.1% N-lauroylsarcosine, 2% Block-
ing Reagent. Membrane was washed twice with 2·
SSC, 1% SDS at 60 ꢁC for 15 min.
4.6. PCR amplification of cDNA
Inosine-containing degenerate oligonucleotide prim-
ers (72S, 119S, 257A, and 318A, the number of primer
indicates the amino acid number of corresponding
A. thaliana AtIPT1) based on the highly conserved se-
quences of A. thaliana AIPTs were used for amplifica-
tion of a core fragment of the cDNA. The sequences
of the primers are as follows: 72S = 50-GG(A/T)
GC(A/C/T) AC(A/C) GGI (A/T)C IGG CAA (A/
G)TC-30, 119S = 50-GT(A/T/G/C) CC(G/T) CA(C/A)
CA(C/A) CT(C/A) CT(C/A) GG-30, 257A = 50-TC(A/
G) AAC TC(C/G/T) GG(A/G/T) AC(A/T) CC(G/T)
AT-30, and 318A = 50-GC(A/G) TC(A/C) ACI C(G/
T)C T(G/T)(A/C/T) ATI T(A/C/T) CCA-30. Nested
PCR was carried out with the primer sets of 72S and
318A, and then with 119S and 257A, to amplify a
431-bp DNA fragment. For the PCR, 30 cycles of
reactions (94 ꢁC for 1 min, 42 ꢁC for 1 min and 72
ꢁC for 1.5 min) were performed each time with a 10
min final extension. The gel-purified PCR product
was ligated into pT7Blue T-Vector (Novagen) and
sequenced.
4.8. RT-PCR analysis
Total cellular RNA prepared from H. lupulus was trea-
ted with RNase-free DNase I at 37 ꢁC for 15 min. RT-
PCR was performed with total RNA (1 or 0.1 lg). The
first Strand cDNA Synthesis Kit for RT-PCR (Roche
Diagnosis) was used for the RT reaction according to
the manufacturerÕs instructions. After incubation with
RNase H for 30 min at 37 ꢁC, 1 ll of the reaction mixture
was used for subsequent PCR using the N-terminal and C-
terminal primers as described above. For the PCR, 30 cy-
cles of reactions (94 ꢁC for 0.5 min, 55 ꢁC for 0.5 min and
72 ꢁC for 2 min) were performed with a 10 min final exten-
sion to amplify the 990-bp DNA fragment.
For the 30-end amplification, a specific primer;
108S = 50-GCT GGA AAA GCC GTG CTG AAA-30,
was designed based on the obtained core sequence.
Then, 30-rapid amplification of cDNA ends (30-RACE)
was carried out with the primer set of 108S and
RACE32 with 30 cycles of reactions (94 ꢁC for 1 min,
49 ꢁC for 1 min and 72 ꢁC for 1.5 min), to amplify an
4.9. Phylogenetic tree
A total of 37 amino acid sequences of isopentenyl-
transferases were aligned and phylogenetic tree was devel-
oped with the CLUSTAL W (1.8) program (DNA Data

Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
2445
Bank of Japan, URL: http://www.ddbj.nig.ac.jp)
4.11. Enzyme purification
(Thompson et al., 1994). Bootstrap resampling (1000
bootstrap, 111 seed) of the original data was used as a
pseudo-empirical test of the reliability of the tree topology
(Felsenstein, 1985; Wu, 1986). The tree was constructed
by use of the majority rule and strict consensus algorithm
implanted in PHYLIP (Felsenstein, 1989). The accession
numbers of GenBank sequence data bank used in the
analysis are as follows; Aquifex aeolicus (AE000721),
Agrobacterium rhizogenes pRiA4 (S06738), A. tumefac-
iens (M83532), A. tumefaciens pTiC58 (AAA27406),
A. vitis pTiS4 (S30106), A. thaliana AtIPT1 (AB061400/
AB062607), A. thaliana AtIPT2 (AB062609), A. thaliana
AtIPT3 (AB061401/AB062610), A. thaliana AtIPT4
(AB061402/AB062611), A. thaliana AtIPT5 (AB061403/
AB062608), A. thaliana AtIPT6 (AB061404/AB062612),
A. thaliana AtIPT7 (AB061405/AB062613), A. thaliana
AtIPT8 (AB061406/AB062614), A. thaliana AtIPT9
(AB062615), Borrelia burgdorferi (AAC67163), Bacillus
subtilis (Z99113), Caenorhabditis elegans (T27538), Chla-
mydia trachomatis (AAC68361), Deinococcus radiodurans
(AAF11245), E. coli (M63655), Homo sapiens
The E. coli cells were harvested by centrifugation,
resuspended in 50 mM potassium phosphate buffer
(KPB), pH 8.0, containing 0.3 M NaCl, and disrupted
on ice by sonication. The cell lysate was centrifuged at
15,000g for 60 min. The supernatant was loaded onto
a column of Ni-NTA resin (Qiagen) which contained
Ni2+ as an affinity ligand. After washing with 50 mM
KPB, pH 6.8, containing 0.3 M NaCl, the recombinant
AIPT was eluted with a gradient of 0–0.5 M imidazole in
the wash buffer. The fraction containing the recombi-
nant IPT was dialyzed with 0.1 M KPB, pH 6.8, con-
taining 1 mM DTT, and stored at ꢀ80 ꢁC. The
purified enzyme preparation gave a single band with
molecular mass of 37 kDa on sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE). Pro-
tein concentration was determined by the Bradford
method (Protein Assay, Bio-Rad) with bovine serum
albumin as standard.
4.12. Enzyme reaction
(AF074918), Lotus japonicus AW720363 , Medicago
*
truncatula AW691664 , Mycobacterium leprae (S72942),
Pseudomonas putida (AF016312), P. solanacearum
(S06739), P. syringae pCK1 (A24937), Rhodococcus fas-
cians pFiD188 (CAA82744), Richettsia prowazekii
The standard reaction mixture contained 1 mM
acceptor substrate, 100 l MDMAPP, and 2.0 lg of
the purified recombinant AIPT in a final volume of
100 ll of 100 mM Tris–HCl buffer, pH 8.0, containing
50 mM KCl, 10 mM MgCl2, 1 mg/ml bovine serum
albumin, 1 mM DTT. Incubations were carried out at
25 ꢁC for 120 min, and stopped by adding 25 ll of
10% AcOH. The products were analyzed by reverse
phase high-performance liquid chromatography
(HPLC) on a TSK-gel ODS-80TS column (4.6 · 150
mm, TOSOH) with a flow rate of 1.0 ml/min. Gradient
elution was performed with 0.85% H3PO4 and CH3CN:
0–15 min, 100% H3PO4; 15–45 min, linear gradient
from 0% to 80% CH3CN. Eluants was monitored at
UV 280 nm.
Steady-state kinetic parameters were determined for
acceptor and donor substrate. The experiments for ki-
netic analyses for acceptor substrate were carried out
in duplicate using five concentrations of AMP (2500,
2000, 1500, 1000 and 500 lM), ADP (100, 80, 60, 40
and 20 lM) or ATP (50, 40, 30, 20 and 10 lM) in the
assay mixture, containing 100 lM DMAPP, 50 mM
KCl, 10 mM MgCl2, 1 mg/ml bovine serum albumin, 1
mM DTT, and the purified recombinant AIPT in a final
volume of 100 ll of 100 mM Tris–HCl buffer, pH 8.0.
The experiments for kinetic analyses for donor substrate
were carried out in duplicate using five concentrations of
DMAPP (50, 40, 30, 20, and 10 lM) in the assay mix-
ture, containing 100 lM ATP, 50 mM KCl, 10 mM
MgCl2, 1 mg/ml bovine serum albumin, 1 mM DTT,
and the purified recombinant AIPT in a final volume
of 100 ll of 100 mM Tris–HCl buffer, pH 8.0. The con-
tent of the purified recombinant AIPT was 4.0 lg for
*
(CAA14962), Solanum tuberosum BE921755 , Saccha-
*
romyces cerevisiae (M15991), Streptomyces coelicolor
(AL022268), Schizosaccharomyces pombe (CAB52278),
Synechocystis sp. PCC6803 (D90911), Thermotoga mari-
tima (C72366). The three EST clones are indicated by
asterisk.
4.10. Cloning and expression of cDNA
A full length cDNA was obtained by RT-PCR using
N-terminal and C-terminal PCR primers; 50-TCT ATC
GGA TCC ATG GAC TAC GCA TCC GTT GC-30
and 50-TGA CCG GGA TCC CTA CTC GTC CAA
GAA GCG AC-30 (the Bam HI site is underlined), with
30 cycles of reactions (94 ꢁC for 0.5 min, 55 ꢁC for 0.5
min and 72 ꢁC for 3 min). The amplified DNA was di-
gested with Bam HI, and cloned into the Bam HI site
of pQE-80L (Qiagen). Thus, the recombinant enzyme
contains an additional hexahistidine tag at the N-termi-
nal. After confirmation of the sequence, the plasmid was
transformed into E. coli M15[pREP4]. The cells harbor-
ing the plasmid were cultured in Luria–Bertani medium
containing 100 lg/ml of ampicillin and 25 lg/ml of
kanamycin at 37 ꢁC until the A600 reached 0.7. Then,
isopropyl-b-
D
-(ꢀ)-thiogalactopyranoside was added to
a final concentration of 1 mM to induce protein expres-
sion, and the culture was incubated further at 18 ꢁC for
24 h.

2446
Y. Sakano et al. / Phytochemistry 65 (2004) 2439–2446
kinetic analysis for AMP, and 2.0 lg for kinetic analysis
for ADP, ATP and DMAPP, respectively. Incubations
were carried out at 25 ꢁC for 30 min, and stopped by
adding 25 ll of 10% AcOH. The reaction products were
analyzed by HPLC, and the molar content of the reac-
tion product was calculated from calibration curve ob-
tained as follows. The same standard reaction as
described above was carried out except that 100 lM
DMAPP was replaced for 0.5 lM [1-3H] DMAPP (1
lCi, 50 pmol) as a donor substrate, and quenched by
adding 25 ll of AcOH–H2O (1:9, v/v). The reaction
products were separated by HPLC monitored at UV
280 nm with five different injection volumes of reaction
solution (5, 10, 15, 20, and 25 ll), the radioactivity in
each fraction was determined by liquid scintillation
counter (Aloka LSC-3100), and the data sets of molar
content and HPLC peak area were plotted to obtain
the calibration curve. Michaelis–Menten plots of data
were employed to derive the apparent KM and kcat val-
ues (average of duplicates standard deviation) using
EnzFitter software (BIOSOFT).
References
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach
using bootstrap. Evolution 39, 783–791.
Felsenstein, J., 1989. PHYLIP-phylogeny inference package (version
3.2). Cladistics 5, 164–166.
Kakimoto, T., 2001. Identification of plant cytokinin biosynthetic
enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyl-
transferases. Plant Cell Physiol. 42, 677–685.
Mok, D.W., Mok, M.C., 2001. Cytokinin metabolism and action.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 89–118.
Morris, R.O., Blevins, D.G., Dietrich, J.T., Durley, R.C., Gelvin, S.B.,
Gray, J., Hommes, N.G., Kaminek, M., Mathews, L.J., Meilan,
R., Reinbott, T.M., Sayavedra-Sato, L., 1993. Cytokinins in plant
pathogenic bacteria and developing cereal grains. Aust. J. Plant
Physiol. 20, 621–637.
Mukai, J., Yamamoto, N., 1995. An isolation method for DNA/RNA
from woody plants. In: Shimamoto, I., Sasaki, T. (Eds.), Protocols
for PCR in Plants. Shujunsha Co. Ltd., Tokyo, Japan, pp. 56–58
(in Japanese).
Murray, M.G., Thompson, W.F., 1980. Rapid isolation of high
molecular weight plant DNA. Nucl. Acids Res. 8, 4321–4325.
Okada, Y., Ito, K., 2001. Cloning and analysis of valerophenone
synthase gene expressed specifically in lupulin gland of hop
(Humulus lupulus L.). Biosci. Biotechnol. Biochem. 65, 150–155.
Okada, Y., Sugimoto, M., Ito, K., 2001. Molecular cloning and
expression of farnesyl pyrophosphate synthase gene responsible for
essential oil biosynthesis in hop (Humulus lupulus). J. Plant Physiol.
158, 1183–1188.
4.13. Site-directed mutagenesis
Asp62 of H. lupulus AIPT was replaced by Ala using
the QuikChangeTM XL Site-Directed Mutagenesis Kit
(Stratagene) and a pair of complementary mutagenic
primers as follows: sense 50-C CCT CTC GAA GTC
ATA AAC TCG GCT AAA ATG CAG G-30, anti sense
50-C CTG CAT TTT AGC CGA GTT TAT GAC TTC
GAG AGG G-30 (mutated base is underlined). The plas-
mid containing D62A mutant was used to transform
competent XL10-Goldꢂ ultracompetent cells. Muta-
genic plasmid DNA was isolated from selected trans-
formants and analyzed by restriction mapping and
DNA sequencing. The verified mutant clones were
transformed into competent cells for expression and iso-
lation as described above.
Paniego, N.B., Zuurbier, K.W., Fung, S.Y., van der Heijden, R.,
Scheffer, J.J., Verpoorte, R., 1999. Phlorisovalerophenone syn-
thase, a novel polyketide synthase from hop (Humulus lupulus L.)
cones. Eur. J. Biochem. 262, 612–616.
Sakakibara, H., Takei, K., 2002. Identification of cytokinin biosyn-
thesis genes in Arabidopsis: a breakthrough for understanding the
metabolic pathway and the regulation in higher plants. Plant Cell
Regul. 21, 17–23.
Stevens, J.F., Taylor, A.W., Nickerson, G.B., Ivancic, M., Henning, J.,
Haunold, A., Deinzer, M.L., 2000. Prenylflavonoid variation in
Humulus lupulus: distribution and taxonomic significance of
xanthogalenol and 40-O-methylxanthohumol. Phytochemistry 53,
759–775.
Sun, J., Niu, Q.-W., Tarkowski, P., Zheng, B., Tarkowska, D.,
Sandberg, G., Chua, N.-H., Zuo, J., 2003. The Arabidopsis
AtIPT8/PGA22 gene encodes an isopentenyl transferase that is
involved in de novo cytokinin biosynthesis. Plant Physiol. 131,
167–176.
Takei, K., Sakakibara, H., Sugiyama, T., 2001. Identification of genes
encoding adenylate isopentenyltransferase, a cytokinin biosynthesis
enzyme, in Arabidopsis thaliana. J. Biol. Chem. 276, 26405–26410.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence align-
ment through sequence weighting, position-specific gap penalties
and weight matrix choice. Nucl. Acids Res. 22, 4673–4680.
Wu, C.F.J., 1986. Jacknife, bootstrap and other sampling plans in
regression analysis. Ann. Statist. 14, 1261–1295.
Acknowledgements
This work was in part supported by the 21st Century
COE Program, and Grant-in-Aid for Scientific Research
(Nos. 1531053 and 16510164) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan,
and by Grant-in-Aid from the Tokyo Biochemical Re-
search Foundation, and The Sumitomo Foundation,
Japan.
Zuurbier, K.W.M., Fung, S.-Y., Scheffer, J.J.C., Verpoorte, R., 1998.
In vitro prenylation of aromatic intermediates in the biosynthesis
of bitter acids in Humulus lupulus. Phytochemistry 49, 2315–2322.