A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline

Article abstract of DOI:10.1080/09168451.2014.882759

A putative indole diterpene biosynthetic gene cluster composed of eight genes was identified in a genome database of Phomopsis amygdali, and from it, biosynthetic genes of fusicoccin A were cloned and characterized. The six genes showed significant similarities to pax genes, which are essential to paxilline biosynthesis in Penicillium paxilli. Recombinants of the three putative prenyltransferase genes in the cluster were overexpressed in Escherichia coli and characterized by means of in vitro experiments. AmyG is perhaps a GGDP synthase. AmyC and AmyD were confirmed to be prenyltransferases catalyzing the transfer of GGDP to IGP and a regular di-prenylation at positions 20 and 21 of paxilline, respectively. AmyD is the first know example of an enzyme with this function. The K_m values for AmyD were calculated to be 7.6 ± 0.5 μM for paxilline and 17.9 ± 1.7 μM for DMAPP at a k_cat of 0.12 ± 0.003/s.

Full text of DOI:10.1080/09168451.2014.882759

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A fungal prenyltransferase catalyzes the regular di-
prenylation at positions 20 and 21 of paxilline
a
a
b
b
a
Chengwei Liu , Motoyoshi Noike , Atsushi Minami , Hideaki Oikawa & Tohru Dairi
a
Graduate School of Engineering, Hokkaido University, Sapporo, Japan
b
Graduate School of Science, Hokkaido University, Sapporo, Japan
Published online: 22 May 2014.
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To cite this article: Chengwei Liu, Motoyoshi Noike, Atsushi Minami, Hideaki Oikawa & Tohru Dairi (2014) A fungal
prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline, Bioscience, Biotechnology, and
Biochemistry, 78:3, 448-454, DOI: 10.1080/09168451.2014.882759
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Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 3, 448–454
A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20
and 21 of paxilline
1
1
2
2
1,
Chengwei Liu , Motoyoshi Noike , Atsushi Minami , Hideaki Oikawa and Tohru Dairi *
1
2
Graduate School of Engineering, Hokkaido University, Sapporo, Japan; Graduate School of Science, Hokkaido
University, Sapporo, Japan
Received October 22, 2013; accepted November 26, 2013
http://dx.doi.org/10.1080/09168451.2014.882759
A putative indole diterpene biosynthetic gene clus-
ter composed of eight genes was identified in a gen-
ome database of Phomopsis amygdali, and from it,
biosynthetic genes of fusicoccin A were cloned and
characterized. The six genes showed significant simi-
larities to pax genes, which are essential to paxilline
biosynthesis in Penicillium paxilli. Recombinants of
the three putative prenyltransferase genes in the
cluster were overexpressed in Escherichia coli and
characterized by means of in vitro experiments.
AmyG is perhaps a GGDP synthase. AmyC and
AmyD were confirmed to be prenyltransferases cat-
alyzing the transfer of GGDP to IGP and a regular
di-prenylation at positions 20 and 21 of paxilline,
respectively. AmyD is the first know example of an
enzyme with this function. The Km values for AmyD
were calculated to be 7.6 ± 0.5 μM for paxilline and
each of the pax genes (paxG, paxC, paxM, paxB, paxP,
and paxQ) by heterologous expression, by in vitro
assay
biotransformation.
with
recombinant
enzymes,
and
by
2
3)
Moreover, the prenylation of
indoles/indole-3-glycerol phosphates by PaxC and step-
wise regular di-prenylation at positions 21 and 22 of
paxilline by PaxD was clearly confirmed by in vitro
2
3,24)
experiments with purified recombinant enzymes.
We have also reported on the function of atmD,
which is located in the aflatrem biosynthetic gene clus-
2
5)
ter in Aspergillus flavus and encodes an enzyme with
32% amino acid identity to PaxD. It catalyzed the
reverse mono-prenylation of paxilline at position 20
and position 21. Both AtmD and PaxD accepted paspa-
line, an intermediate of paxilline biosynthesis that has a
structure similar to paxilline, but the regiospecificity
and the regular/reverse specificity for prenylation were
unexpectedly altered. Both AtmD and PaxD catalyzed
the regular mono-prenylation of paspaline at position
1
7.9 ± 1.7 μM for DMAPP at a kcat of 0.12 ± 0.003/s.
2
6)
Key words; indole diterpene; paxilline; fungi; Phom-
opsis amygdali; prenyltransferases
21 and position 22. These results suggest that fungal
indole diterpene prenyltransferases have the potential to
alter regio- and regular/reverse specificities for prenyla-
tion and are applicable in the synthesis of industrially
useful compounds.
The isoprenoid compounds found in nature to date,
at more than 50,000 known examples, include industri-
ally useful compounds such as flavors, antibiotics, and
As part of our survey of a new fungal indole diter-
pene prenyltransferase, we identified a putative indole
diterpene biosynthetic gene cluster composed of eight
genes in the genome database of Phomopsis amygdali
1
–3)
plant hormones, among others.
In some cases, iso-
prenoids are attached to other moieties, such as a poly-
4
)
5)
6)
27)
ketide, indole/tryptophan, or (iso)flavonoid, or to
(Fig. 1(A)), a diterpene glucoside fusicoccin A pro-
7
,8)
phenazine moieties.
Prenylated indole alkaloids such
ducer, for which we had identified the biosynthetic
genes. The six genes (amyG, amyC, amyM, amyB,
amyP, and amyQ) have significant similarities to the
9
)
10)
11,12)
as paspalitrem A, aflatrem, janthitrems,
sheari-
1
3,14)
15)
16)
17)
nines,
penitrem A, lolilline, and lolitrems,
2
2)
all of which are produced by filamentous fungi, are
corresponding pax genes,
which are essential for
1
8)
examples of this. These compounds are derived from
polyprenyl diphosphates and tryptophan or its deriva-
tives, and they show diverse chemical structures and
paxilline biosynthesis. The remaining two genes are
similar to prenyltransferases (amyD) and cytochrome
P450s, suggesting that the cluster participates in the
biosynthesis of a prenylated- and oxidized-paxilline
such as lolilline and lolitrem. In this study, we investi-
gated the substrate specificities of AmyD together with
the PaxG and PaxC orthologs (AmyG and AmyC) with
recombinant enzymes.
1
9–21)
biological activities.
Pioneering studies of biosynthetic genes and
enzymes for indole-diterpenes were carried out by Scott
2
2)
et al. with Penicillium paxilli, a paxilline producer.
Recently, we characterized details of the functioning of
*Corresponding author. Email: dairi@eng.hokudai.ac.jp
Abbreviations: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GGDP, geranylgeranyl diphosphate; FDP, farnesyl diphosphate;
GDP, geranyldiphosphate; IGP, indole-3-glycerol phosphate.
©
2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

Regular di-prenylation at positions 20 and 21 of paxilline
449
Fig. 1. The silent indole diterpene biosynthetic gene cluster identified in P. amygdali.
Note: Gene organization of amyD, amyC, and amyG (A), SDS-PAGE analysis of their recombinants (B), and gel filtration chromatography of
recombinant AmyD (C). In (C), elution profiles of the standard proteins, aldolase (a, 158 kDa), albumin (b, 67 kDa), chymotrypsinogen A (c, 25
kDa), and ribonuclease A (d, 13.7 kDa); top and purified AmyD (bottom, e) are shown.
Materials and methods
Overexpression and purification of prenyltrans-
ferases. After subcloning and sequence confirmation,
the cDNA carrying the amyC, amyD, or amyG gene
was inserted into the pMAL-c2X and into pMAL-c5X.
In the resulting plasmids, pMAL-c2X-AmyC, pMAL-
c5X-AmyD, and pMAL-c2X-AmyG, the recombinant
protein was expressed as an N-terminal MBP-fused
protein. The expression and purification conditions for
the recombinant enzymes were essentially the same as
those described in the manufacturer’s protocols. In
brief, E. coli TB1 (New England Biolad, Japan,
Tokyo), harboring the expression plasmid, was grown
at 37 °C in Luria Broth medium with ampicillin
General. Sequence analysis of the PCR fragments
was performed by the dideoxy chain termination
method with an automatic DNA sequencer (Li-Cor,
Model 4000L, NE). Cell disruption was done with an
Ultrasonic Disruptor (TOMY, UD-200, Tokyo). Analy-
sis of the samples during protein purification was done
by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE), and the proteins were visualized by Coomassie
brilliant blue staining. The protein concentration was
determined by the Bradford method, with bovine serum
albumin as a standard. Plasmids from Escherichia coli
were prepared with a Qiagen plasmid kit. All restriction
enzymes, T4 ligase, and calf intestinal alkaline phos-
phatase were purchased from Toyobo (Osaka, Japan)
and were used following the manufacturer’s instruc-
tions. Transformation of E. coli with plasmid DNA by
electroporation was performed under standard condi-
tions with a Gene Pulser electroporation system (Bio-
Lad Japan, Tokyo).
(100 μg/mL) and D-glucose (0.2%). Expression of the
recombinant protein was induced by adding 1 mM
IPTG when the optical density at 600 nm reached about
0.8, and cultivation was continued for an additional 16
h at 18 °C. The recombinant protein was purified by
amylose affinity column chromatography (New Eng-
land Biolabs). After elution of the recombinant
enzymes, purity was analyzed by SDS-PAGE on 10%
gels. The protein concentration was determined by pro-
tein-dye standard assay (Bio-Rad) with bovine serum
albumin as standard. For determination of the molecu-
lar mass and subunit structure, the purified enzyme was
loaded onto a HiLoad 16/60 Superdex 75 pg gel-filtra-
tion column (Amersham Biosciences, Piscataway, NJ)
and eluted with a buffer containing 50 mM Tris–HCl
(pH 8.0) and 10 mM NaCl. The retention volume of
AmyD was compared to those of the marker proteins.
The markers applied to the column were aldolase (158
kDa), albumin (67 kDa), chymotrypsinogen A (25
kDa), and ribonuclease A (13.7 kDa) (GE Healthcare,
Little Chalfont, UK).
cDNA preparation by heterologous expression in
Aspergillus oryzae. The coding regions, including the
intron sequences of the amyC, amyD, and amyG genes,
2
8)
were amplified by PCR with P. amygdali Niigata-2
genomic DNA and a primer set, as shown in
Supplemental Table 1. The amplified DNA fragments
were digested with the appropriate restriction enzymes
and inserted into the corresponding sites of the expres-
2
9)
sion vector, pTAex3,
to create pTAex3-amyG,
pTAex3-amyC, and pTAex3-amyD. Then, each of the
2
3)
plasmids was introduced into A. oryzae. Mycelia of
the transformants were inoculated into a medium (3.5%
Czapek-Dox, 2% starch, 1% polypeptone; final volume
In vitro assays of prenyltransferases.
The assay
1
00 mL) and cultivated at 30 °C for 3 d in a reciprocal
mixture of AmyG in a final volume of 100 μL con-
shaker (125 strokes/min). cDNA preparation was
performed by a method described previously,
the primer set shown in Supplemental Table 1.
tained 0.5 mM of allylic substrates, 0.5 mM of IPP, 50
3
0)
with
2+
mM Tris–HCl (pH 8.0), 5 mM Mg and a suitable
amount of AmyG. The mixture was incubated at 30 °C

450
C. Liu et al.
overnight. After centrifugation at 15,000 rpm for 30
min, the precipitated products were solubilized with
43.2, 49.5, 50.4, 72.5, 72.6, 77.6, 83.3, 109.4, 116.9,
119.6, 122.9, 124.4, 124.7, 125.3, 130.4 (x2), 130.6,
1
00 μL of water and analyzed by high performance
130.9, 138.4, 151.1, 168.2, 199.3. High resolution
+
liquid chromatography (HPLC). The analytical condi-
tions were as follows: Merck Mightisil RP-18GP Aqua
column (250 mM × 4.6 mM) (Kanto Chemicals, Tokyo);
mobile phase of acetonitrile in 10 mM potassium phos-
phate (pH 7.4) and 0.5% ion-pair reagents (0.5 mol/L,
tetrabutylammonium dihydrogen phosphate solution;
Wako, Osaka, Japan) (0 to 15 min, 40% acetonitrile;
(HR)-ESIMS: calcd. for C H NO
[M + H] :
3
7
50
4
572.3734, observed: 572.3775.
Accession numbers.
The nucleotide sequences
reported here have been submitted to the DDBJ/Gen-
BankTM/EBI Data Bank under Accession Nos.
AB839408
(amyG),
AB839409
(amyC),
and
1
1
5–30 min, 40–100%; 30–45 min, 100%); flow rate,
.0 mL/min; detection, 205 nm. The assay methods for
AB839410 (amyD).
AmyC and AmyD were essentially as previously
2
3,24,26)
reported.
Results
The steady-state kinetic parameters of AmyD were
determined by fitting to the Michaelis–Menten equa-
tion. The assay was linear with respect to the protein
concentration at up to 1 μg for 30 min of incubation,
and no substrate inhibition was observed for paxilline
or DMAPP at up to 1.0 mM of a given substrate. The
assays to determine the kinetic parameters of paxilline
in a final volume of 100 μL contained 50 mM Tris–
HCl (pH 8.0), 0.5 mM DMAPP, 0.2 μg of AmyD, and
Attempt to identify a compound governed by the
cluster
Although the predicted amino acid sequences of
AmyG and AmyC were similar to PaxG and PaxC,
respectively, that of AmyD showed lower similarity to
PaxD/AtmD (see below), and we were not sure
whether the intrinsic substrate of AmyD is paxilline or
another compound. In an attempt to identify the sub-
strate of AmyD, we searched for an indole diterpene
compound in the culture broth of P. amygdali, but no
indole diterpene-related compounds were detected by
LC-ESI-MS analysis under standard cultivation condi-
tions. Trials to identify a compound related to indole
diterpene by varying the culture medium and condi-
1
μM to 0.5 mM paxilline. The mixture was incubated
at 30 °C for 20 min. When the concentration of paxil-
line held steady at 0.25 mM, the concentration of
DMAPP was varied from 5 μM to 0.5 mM.
3
1)
Liquid chromatography–electrospray ionization mass
spectrometry (LC/ESI-MS) analysis. Products formed
in the in vitro assays were analyzed by LC/ESI-MS
tions
tors,
and by adding histone deacetylase-inhibi-
which are known to induce secondary
3
2,33)
metabolite genes, also failed. Next, an engineered strain
from which the gene responsible for the first biosyn-
thetic reaction of fusicoccin A (pafs gene) was deleted
(Supplemental Fig. 3) to supply more IPP for indole
diterpene biosynthesis was constructed because
P. amygdali produced large amounts (>1 g/L) of
fusicoccin-related compounds. That strain, however,
produced no specific compounds. Then, we focused on
a transcriptional regulator, since the activation of
(
Waters Acquity UPLC equipped with SQD2, Waters).
The analytical conditions were as follows: Waters Ac-
quity UPLC BEH C18 1.7 μm column (2.1 × 50 mM);
column temperature, 40 °C; detection, positive mode;
mobile phase, 0.1% formic acid: acetonitrile = 30:70 at
3
4)
5
3
min, and a linear gradient to 50:50 for an additional
0 min; flow rate, 0.3 mL/min; cone voltage, 30 V.
transcriptional regulators sometimes results in high pro-
Metal dependency of PAPT.
Divalent metal ions
35,36)
duction of secondary metabolites in fungi,
but no
2
+
2+
2+
2+
2+
2+
2+
(
5 mM of Mg , Ca , Fe , Cu , Zn , Mn , Ni ,
candidate genes were located at the flanking regions of
the cluster.
2
+
and Co ) or 5 mM EDTA was added to the standard
reaction mixture.
Preparation of cDNA by heterologous expression
and overexpression of recombinant enzymes
Structural analysis of the reaction product.
The
reaction product formed from paxilline and DMAPP by
AmyD was fractionated by a preparative HPLC. The
Since we could not estimate the intrinsic substrate of
AmyD, we attempted to prepare cDNAs by heterolo-
1
13
H- and C-NMR spectra were recorded on a Bruker
AMX-500 spectrometer: (Supplemental Table S2).
NMR δH (CDCl , 500 MHz) (Supplemental Fig. 1)
37)
gous expression of it in A. oryzae and to examine
the function of the recombinant enzyme with paxilline
as substrate. The cDNAs of amyC and amyG were also
prepared by the same method. Each amyC, amyD, and
amyG gene was amplified by PCR and introduced into
pTAex3, in which the inserted gene was expressed
under the control of the promoter of the α-amylase
3
1
1
1
2
1
2
.04 (s, 3H), 1.28 (s, 3H), 1.30 (s, 3H), 1.32 (s, 3H),
.46 (m, 1H), 1.64 (m, 1H), 1.68 (s, 3H), 1.70 (s, 3H),
.72 (s, 3H), 1.78 (m, 1H), 1.80 (s, 3H), 1.90 (m, 1H),
.04 (m, 1H), 2.07 (m, 1H), 2.32 (m, 1H), 2.57 (m,
H), 2.79 (m, 1H), 2.85 (m, 1H), 3.36 (d, J = 6.8 Hz,
H), 3.61 (m, 2H), 3.73 (d, J = 2.0 Hz, 1H), 4.86 (brt,
29)
gene of A. oryzae. The transformants were cultivated,
and then, cDNAs were prepared. The predicted gene
products of AmyG, AmyC, and AmyD consisted of
J = 9.7 Hz, 1H), 5.18 (m, 1H), 5.27 (m, 1H), 5.89 (d, J
1.7 Hz 1H), 6.91 (d, J = 8.3 Hz 1H), 7.09 (d, J = 8.3
Hz 1H), 7.65 (s, 1H). NMR δC (CDCl , 125 MHz)
=
339, 347, and 457 amino acids, respectively. The
3
enzymes most closely homologous to AmyG and
AmyC were PaxG (32% amino acid identity) and PaxC
(
2
Supplemental Fig. 2) 16.2, 17.9, 18.2, 19.7, 20.9,
4.2, 25.7, 25.8, 26.6, 28.0, 28.5, 29.1 (x2), 31.1, 34.4,
(40%), respectively, suggesting that the cluster

Regular di-prenylation at positions 20 and 21 of paxilline
451
participates in indole diterpene biosynthesis, but AmyD
showed only low similarity to PaxD (13% amino acid
identity), a prenyltransferase the gene locates in the
paxilline biosynthetic gene cluster.
Each of the cDNAs was cloned into pMAL-c2X and
pMAL-c5X vector for protein expression in E. coli.
Maltose binding protein-fused recombinant enzymes
were expressed successfully in soluble form under the
conditions described above in “Materials and Meth-
ods”. The purified enzymes were subjected to SDS-
PAGE to confirm their molecular sizes and purity
(
Fig. 1(B)). The recombinant enzyme of AmyD was
then subjected to gel filtration to estimate its subunit
structure. As shown in Fig. 1(C), a peak with a calcu-
lated molecular mass of 104 kDa was detected, suggest-
ing that AmyD forms a monomer structure different
from recombinant PaxD and AtmD, both of which
showed homodimer structures, but this result might
have been due to fusion of the large MBP at N-termi-
Fig. 3. HPLC profiles of the AmyC reaction products.
Note: (A) with indole; (B) with IGP; (C) geranylgeranyl indole
standard.
3
8)
nus, which is known to form a monomer structure.
Functional analysis of AmyG and AmyC
slightly accepted, in the same manner as PaxC
Catalytic activity was then examined with purified
recombinant enzymes. Since AmyG was very similar to
PaxG, the assay used for PaxG was employed. After
recombinant AmyG was incubated with IPP and
DMAPP, GDP, or FDP, the reaction product was ana-
lyzed by HPLC. When FDP and GDP were used, a
peak that eluted at the same retention time as that of
standard GGDP was detected together with another
peak, which was farnesylgeranyl diphosphate (C25) in
view of the retention times of FDP (C15) and GGDP,
as shown in Fig. 2(A) and (B), but DMAPP was not
utilized (data not shown). Considering that amyG clus-
ters with many orthologs of the paxilline biosynthetic
genes (Fig. 1(A) and Supplemental Table 3) and that
the chain length of the product generated by the
in vitro assay with GGDP synthase is known to vary
(
Fig. 3(A)). Although PaxC had been found to catalyze
the transfer of FDP to IGP, yielding farnesyl indole,
AmyC did not accept FDP (data not shown).
Functional analysis of AmyD
We characterized the recombinant AmyD using pax-
illine as substrate. The recombinant AmyD was incu-
bated with paxilline and DMAPP and the reaction
products were analyzed by HPLC, and a major product
was specifically detected (Fig. 4(A)). Selected ion chro-
matograms obtained by LC/ESI-MS analysis showed a
specific peak with a molecular mass corresponding to
di-prenylated paxilline (Fig. 4(C) and (D)). HR (high
resolution)-ESIMS of the product indicated the molecu-
lar formula C H NO , confirming the production of
di-prenylated paxilline. The H-NMR spectra showed
the characteristic signals of regular prenyl moieties:
two olefinic protons, 5.18 (m, 1H) and 5.27 ppm (m,
1
3
3
7
49
4
3
9)
1
depending on reaction conditions,
haps a GGDP synthase.
AmyG was per-
The enzymatic activity of AmyC was examined by
2
3)
the assay method used for PaxC.
Geranylgeranyl
H); two allylic methylene protons, 3.61 (m, 2H) and
.36 (d, J = 6.8 Hz, 2H); and for the 4,5-di-substituted
indole formed efficiently when AmyC was incubated
with IGP and GGDP (Fig. 3(B)). Indole was also
indole signals, aromatic protons, 6.91 (d, J = 8.3 Hz,
H) and 7.09 ppm (d, J = 8.3 Hz, 1H)], indicating that
1
the substitution of the prenyl moieties at the C-20 and
C-21 positions on the indole moiety. Finally, extensive
NMR data analysis, including hetero-nuclear single
quantum coherence (HSQC) (Supplemental Fig. 4), het-
ero-nuclear multiple quantum coherence (HMBC) (Sup-
plemental Fig. 5), correlation spectroscopy (COSY)
(Supplemental Fig. 6), and nuclear overhauser effect
spectroscopy (NOESY) (Supplemental Fig. 7) indicate
that the structure was 20,21-di-prenylated paxilline
(Supplemental Fig. 8). To the best of our knowledge,
AmyD is the first enzyme known to catalyze the
di-prenylation of paxilline at positions 20 and 21.
Next, we tested paspaline, an intermediate of paxil-
line biosynthesis that has a structure similar to paxil-
line, as substrate, because both PaxD and AtmD have
been found to accept this compound. The reaction
products were analyzed by HPLC, and three small
peaks were detected. By LC/ESI-MS analysis, one peak
Fig. 2. HPLC profiles of AmyG reaction products.
Note: (A) with IPP and FDP; (B) with IPP and GDP; C, GGDP
standard.

452
C. Liu et al.
Fig. 4. HPLC and LC/ESI-MS analysis of the products formed in the reaction of paxilline and DMAPP catalyzed by AmyD.
Note: The reaction products formed with (A, C) and without (B) AmyD were analyzed by HPLC (A, B) and LC/ESI-MS (C, D). Selected ion
chromatograms (C) and spectra of the peak (D) are shown.
was perhaps a di-prenylated paspaline, and the other
two were mono-prenylated paspalines (Supplemental
Fig. 9). We could not determine the structure of these
compounds due to low yield. However, the following
observations suggested that the latter product might be
about 7.0 (Supplemental Figs. 11 and 12). Since some
prokaryotic prenyltransferases require divalent cations
for their activity, the effects and dependence of divalent
metal ions on AmyD activity were tested (Supplemen-
2
+
2+
tal Fig. 13). The addition of Mg and Ca enhanced
enzymatic activity in contrast to EDTA, which
decreased the activity to less than 50% of control.
2
0-, 21-, or 22-mono-prenylated paspaline. The reten-
tion time of one of the mono-prenylated paspalines
Supplemental Fig. 9A, peak B) was the same as that
2
+
2+
(
Activity was lost when Cu and Zn were added.
The kinetic parameters of AmyD were also investi-
gated (Supplemental Fig. 14). The enzyme reaction fol-
lowed Michaelis–Menten kinetics. By means of Hanes–
Woolf plots, the Km values were determined to be 7.6
± 0.5 μM for paxilline and 17.9 ± 1.7 μM for DMAPP.
The kcat value was 0.12 ± 0.003/s.
of the 21- or 22-mono-prenylated paspaline, which has
been found to form the same substrate by both AtmD
2
6)
and PaxD. Both compounds were eluted at the same
retention time by conventional reverse-phase column.
The product formed by AmyD was also found to be a
mixture of the 21- and 22-mono-prenylated paspaline
by XBridgeTM Phenyl Column analysis (Supplemental
Fig. 10). Since we did not access the standard com-
pound of 20-mono-prenylated paspaline, we could not
examine the retention time of this compound by the
XBridgeTM Phenyl Column analysis. Considering that
AmyD catalyzed prenylation at the 20 and 21 positions
rather than the 21 and 22 positions, prenylation at the
Discussion
We identified a putative indole diterpene biosyn-
thetic gene cluster composed of eight genes in the
genome database of P. amygdali (Fig. 1(A)), from the
strain we cloned the biosynthetic genes of fusicoccin
2
0 position was also highly probable.
3
4)
A. We attempted to identify a compound governed
by the cluster, but all attempts failed. The following
results, however, suggest that the cluster might be
responsible for the biosynthesis of a lolilline/lolitrem
type of compound. The six genes (amyG, amyC,
amyM, amyB, amyP, and amyQ) have significant
Biochemical characterization of AmyD
The biochemical properties of AmyD were investi-
gated with paxilline and DMAPP as substrates. The
optimal temperature and pH values were 55 °C and

Regular di-prenylation at positions 20 and 21 of paxilline
453
Fig. 5. Summary of the reactions catalyzed by AmyC, D, and G.
2
2)
similarities to the corresponding pax genes,
which
Acknowledgments
are essential for paxilline biosynthesis. Indeed, AmyG
and AmyC were perhaps a GGDP synthase and a ger-
anylgeranyl indole synthase, respectively, both of
which are essential enzymes in the biosynthesis of
indole diterpene-related compounds (Fig. 5). More-
over, AmyD showed 58% amino acid identity to the
product of ltmE, which has been identified in the lolit-
rems biosynthetic gene cluster in Epichloë festucae, in
contrast its low similarity to AtmD and PaxD (17%
and 13%). Scott et al. examined the function of the
ltmE gene by constructing a gene-deletion mutant,
introducing the mutant into its host plant, and per-
forming structural analysis of a lolitrem-related com-
pound produced by the endophyte. They suggested
that LtmE catalyzes the di-prenylation of the A-ring
of terpendole I, a paxilline-related compound, at posi-
This study was supported by Grants-in-Aid for Sci-
entific Research (23108101 to T. Dairi and 22108002
to H. Oikawa) from the Japan Society for the Promo-
tion of Science (JSPS).
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