Identification of Chimeric αβγ Diterpene Synthases Possessing both Type II Terpene Cyclase and Prenyltransferase Activities

Article abstract of DOI:10.1002/cbic.201700445

Two unusual diterpene synthases composed of three domains (α, β, and γ) were identified from fungal Penicillium species. They are the first enzymes found to possess both type II terpene cyclase (TC) and prenyltransferase (PT) activities. These enzymes were characterized by heterologous expression in Aspergillus oryzae and in vitro experiments with wild-type, mutated, and truncated enzymes. The results revealed that the α domain in the C-terminal region of these enzymes was responsible for the PT activity, whereas the βγ domains in the N-terminal region composed the type II TC, and formed copalyl diphosphate (2). Additionally, between the α and βγ domains, there is a characteristic linker region, in which minimal secondary structure is predicted. This linker does not exist in the characterized three-domain (αβγ) terpene synthases known as monofunctional type I or type II TCs, or bifunctional type I and type II TC enzymes. Therefore, both the catalytic activities and protein architecture substantially differentiate these new enzymes from the previously characterized terpene synthases.

Full text of DOI:10.1002/cbic.201700445

Accepted Article
Title: Identification of Chimeric αβγ Diterpene Synthases Possessing
both Type II Terpene Cyclase and Prenyltransferase Activities
Authors: Ikuro Abe, Takaaki Mitsuhashi, and Masahiro Okada
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201700445
Link to VoR: http://dx.doi.org/10.1002/cbic.201700445
A Journal of
www.chembiochem.org

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
Identification of Chimeric αβγ Diterpene Synthases Possessing
both Type II Terpene Cyclase and Prenyltransferase Activities
Takaaki Mitsuhashi[a], Masahiro Okada[a], and Ikuro Abe*[a]
Abstract: We identified two unusual diterpene synthases composed
of three domains (α, β, and γ) from fungal Penicillium species. They
are the first enzymes found to possess both Type II terpene cyclase
(TC) and prenyltransferase (PT) activities. These enzymes were
characterized by heterologous expression in Aspergillus oryzae and
in vitro experiments with wild-type, mutated, and truncated enzymes.
The results revealed that the α domain in the C-terminal region of
these enzymes was responsible for the PT activity, while the βγ
domains in the N-terminal region composed the Type II TC, and
formed copalyl diphosphate 2. Additionally, between the α and βγ
domains, there is a characteristic linker region in which minimal
secondary structure is predicted. This linker does not exist in the
characterized three domain (αβγ) terpene synthases known as the
mono functional Type I or Type II TCs, or the bifunctional Type I TC
+ Type II TC enzymes. Therefore, both the catalytic activities and
protein architecture substantially differentiate these new enzymes
from the previously characterized terpene synthases.
composed of αβ or αβγ domains. The Type II TCs are enzymes
with either βγ or αβγ domains.
Indeed, some TCs and PTs are bifunctional enzymes
fused with another domain that constitutes a distinct active site
for successive reactions.[6] For example, the bifunctional αβγ
TCs, found in plants and fungi, can catalyze tandem Type II and
Type I TC reactions.[7] In this case, the Type I TC active site is in
the α domain, and the Type II TC active site is at the interface of
the βγ domains. Another example of bifunctional catalysis is a
group of di- and sesterterpene synthases from fungi, composed
of αα domains. The N-terminal α domain works as a Type I TC,
while the C-terminal α domain is a PT that forms a substrate
used by the N-terminal Type I TC domain.[8] Other αα terpene
synthases were found in several Gram-positive soil bacteria.[9]
The N-terminal α domain catalyzes the Type I TC reaction to
form a cyclized sesquiterpenoid, and uniquely, the C-terminal α
domain is responsible for the protonation, cyclization, and
fragmentation of the cyclized product. Actually, the fusion of the
TC or PT domain with another domain, which catalyzes
Prenyltransferase (PT) and terpene cyclase (TC) are key
enzymes in the formation of the basic carbon skeleton of
terpenoids, which constitute one of the most structurally and
stereochemically diverse families of natural products with more
than 75,000 members. Despite the vast chemodiversity of the
terpenoids, they all originate from simple C5 substrates,
sequential reactions, may provide
a catalytic advantage,
because the physical proximity of the two active sites can
enhance product flux.[10] For the αα di- and sesterterpene
synthases composed of a Type I TC and a PT, this catalytic
advantage seems to be enhanced through an agglomeration
effect,[11] considering that one of them, fusicoccadiene synthase
from Phomopsis amygdali, proved to be a hexamer.[12]
dimethylallyl
diphosphate
(DMAPP)
and
isopentenyl
diphosphate (IPP).[1] In the biosynthesis of terpenoids, the PTs
catalyze a chain elongation reaction utilizing DMAPP and IPP to
form linear isoprenoid precursors, such as (C10) geranyl
diphosphate (GPP), (C15) farnesyl diphosphate (FPP), (C20)
geranylgeranyl diphosphate (GGPP), and (C25) geranylfarnesyl
diphosphate (GFPP).[2] TCs are the enzymes responsible for the
cyclization of the linear isoprenoid precursors to form the
complex carbon skeletons of various cyclic terpenoids.[1, 3] The
TCs are categorized into two distinct classes (Type I and Type
II), depending on their strategies to initiate catalysis. The Type I
TCs initiate cyclization by heterolytic cleavage of the
diphosphate moiety, which generates a reactive carbocation
intermediate. The Type II TCs produce the carbocation
intermediate by the protonation of a C=C double bond or an
epoxide ring. Based on prior structural and mechanistic studies
of PTs and TCs, various combinations of the three basic
domains (α, β, and γ) form the structures of these enzymes.[4]
Typically, both the PTs and microbial Type I TCs contain only an
α domain. This structural homology between PTs and microbial
Type I TCs suggests that they may share common evolutionary
Despite the existence of chimeric Type I TC
+ PT
enzymes[8] and their catalytic advantage, Type II TC + PT
terpene synthases have not been identified so far. Here, we
report two unusual αβγ diterpene synthases, as the first
bifunctional proteins consisting of a Type II TC and a PT.
Characteristically, the amino acid sequences of these enzymes
suggest that a linker region exists between the α domain and the
βγ domains. This type of linker region is not present in the other
αβγ terpene synthases.[1, 3] These enzymes were characterized
by heterologous expression in Aspergillus oryzae and in vitro
experiments with wild-type, mutated, and truncated enzymes.
We searched for Type II TC + PT enzymes by a genome-
based approach. We tried to find a gene encoding a chimeric
protein composed of a Type II TC homolog and a PT homolog.
This attempt afforded two candidates in fungi. One was a 963
amino acid residue protein from Penicillium verruculosum
TPU1311[13] (accession: LC316181). The other was from
Penicillium fellutanum ATCC 48694, and contained 935 amino
acid residues (JGI Protein ID: 403578). Based on their amino
acid sequences, the βγ domain architecture for the Type II TC is
predicted in the N-terminal regions of these enzymes (aa 1-539
and 1-538, respectively), because of the ~25% sequence
origins.[5] In contrast, the plant Type
I TCs are normally
[a]
T. Mitsuhashi, Dr. M. Okada, Prof. Dr. I. Abe
Graduate School of Pharmaceutical Sciences, The University of
Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
E-mail: abei@mol.f.u-tokyo.ac.jp
Supporting information for this article is given via a link at the end of
the document
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
Figure 1. Secondary structure predicted in the amino acid sequences of PvCPS and PfCPS. The α helix and the β strand are shown as blue and orange boxes,
respectively. The linker region is inserted between the N-terminal βγ domain and the C-terminal α domain.
identity and ~40% sequence similarity with PtmT2 from
Streptomyces platensis CB00739,[14] which is a Type II TC with
the βγ domain architecture (Protein Data Bank accession code:
5BP8, Figure S1).[15] In contrast, the C-terminal regions of these
enzymes (aa 651-963 and 609-935, respectively) exhibited
~40% sequence identity and ~60% sequence similarity with the
PT domain of fusicoccadiene synthase from Phomopsis
amygdali,[8h] which forms an α domain architecture (Protein Data
Bank accession codes: 5ERN and 5ERO, Figure S1).[12] Thus,
the C-terminal regions seem to be the α domain for the PT.
Remarkably, the N-terminal Type II TC (βγ) domain and the C-
terminal PT (α) domain are separated by linker regions (aa 540-
650 and 539-608, respectively), which are not found in the
typical αβγ terpene synthases.[3] Minimal secondary structure is
predicted to exist in the linker regions, based on an analysis of
the amino acid sequences (Figure 1). Contrarily, in the typical
αβγ terpene synthases, the α and βγ domains are connected by
an alpha helix straddling these domains, according to prior X-ray
crystal structural analyses (Figure S2).[16] Notably, between
these two unusual αβγ enzymes, the amino acid sequences of
the linkers exhibit relatively low similarity (Figure S3), as
compared with the high homology in the N- and C-terminal
domains (61% sequence identity and 73% sequence similarity
for N-terminal βγ domains, and 74% sequence identity and 88%
sequence similarity for C-terminal α domains). The insertion of a
linker between two distinctive active sites is also found in the αα
terpene synthases.[8, 9] The similarity of the linkers is also low
among the αα enzymes consisting of the Type I TC and PT
domains (Figure S4 and Table S4).
+22.3 (c 1.00, CHCl3), respectively), and a comparison with a
previous report[19] revealed that both metabolites are the same
compound, (+)-copalol 1. This investigation indicated that these
enzymes are responsible for the formation of copalyl
diphosphate 2, and an endogenous phosphatase in A. oryzae
seemed to convert 2 to 1 (Scheme 1). Such dephosphorylation
has previously been reported when another kind of copalyl
diphosphate synthase was introduced into an Escherichia coli
heterologous expression system.[20] Therefore, these enzymes
were designated as Penicillium verruculosum copalyl
diphosphate synthase (PvCPS) and Penicillium fellutanum
copalyl diphosphate synthase (PfCPS).
OPP
OPP
DMAPP
PT domain
× 3
γ
OPP
α
β
IPP
GGPP
OPP
OH
Type ll TC
domain
H
phosphatase
H
γ
α
β
H
H
2
1
To characterize these two unusual αβγ enzymes, at first,
we determined the structures of their products. For this purpose,
Scheme 1. The reactions catalyzed by PvCPS and PfCPS. They catalyze the
same reaction. The domains (α or βγ) responsible for each reaction are
highlighted in red.
they were heterologously expressed in A. oryzae NSAR1,[17]
a
quadruple auxotrophic mutant strain that has been successfully
utilized for biosynthetic studies of fungal natural products.[18] The
mycelial extract of each A. oryzae transformant was analyzed by
GC-MS, and consequently, both transformants produced new
metabolites with m/z 290 [M]+. These metabolites were expected
to be a diterpene alcohol and exhibited the same retention times
and mass spectra (Figure S5). After their isolation, the NMR
analysis (Tables S2 to S3 and Figures S10 to S21), the optical
rotation determination ([α]23 +25.4 (c 0.33, CHCl3) and [α]31
For more detailed characterization, in vitro experiments
with the purified enzymes were performed. First, the wild-type
enzymes were incubated with the C5 substrates, DMAPP and
IPP. After the incubation, the reaction products were hydrolyzed
by a phosphatase, and then analyzed by GC-MS. The detection
of 1 indicated that PvCPS and PfCPS exhibit both Type II TC
and PT activities (Scheme 1, Figure 2A). Additionally, these
D
D
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
enzymes could also utilize GPP+IPP, FPP+IPP, and GGPP as
substrates to produce 2 (Figures S6 to S7). However, GFPP, a
C25 substrate, was not converted to a cyclized sesterterpenene
(data not shown). Thus, the TC domains of these enzymes
proved to be C20-specific cyclases.
terminus truncated mutants of PvCPS and PfCPS do not overlap.
Indeed, aa 560-600 of PvCPS and aa 549-587 of PfCPS are not
necessary for both the TC and PT activities (Figures 2B and 2C).
Thus, for PvCPS and PfCPS, the degrees of the
interdependence between the α domain and the βγ domains are
different from those of the previously characterized αβγ terpene
synthases.
Next, site-directed mutagenesis was performed to
determine whether the N- and C-terminal domains of these
enzymes are actually responsible for the Type II TC and PT
activities, respectively. In general, Type II TCs and PTs contain
characteristic aspartate-rich motifs necessary for the catalytic
activity and constituting part of the active site.[21] The Type II TCs
possess a DXDD motif, in which the central aspartic acid serves
as a general acid to initiate cyclization.[21a] The PTs contain two
DDXX(D,N) motifs involved in substrate binding and catalysis,
via chelation with Mg2+.[21b] In fact, the DXDD motif is present in
the N-terminal domains of PvCPS and PfCPS (D311ADD314 and
D313ADD316, respectively). Meanwhile, each enzyme possesses
two DDXX(D,N) motifs in the C-terminal domain (D727DIQD731
and D851DYLN855 in PvCPS, and D699DIED703 and D823DYLN827 in
PfCPS). To examine the role of the DXDD motif in the N-
terminal domain, the central aspartate residue was substituted
with the nonpolar residue, alanine: D313A for PvCPS and
D315A for PfCPS. These mutants lacked the Type II TC activity
but retained the PT activity, producing GGPP. Conversely,
mutations in the C-terminal aspartate-rich motif (D727A for
PvCPS and D699A for PfCPS) resulted in the loss of the PT
activity; however, these mutants still exhibited the Type II TC
activity, converting GGPP into 2. Therefore, these analyses
indicated that the N-terminal domain is a Type II TC domain, and
the C-terminal domain is a PT domain (Figures 2B and 2C and
Figures S8 to S9).
Truncation variants of these enzymes were prepared, to
determine whether the Type II TC and PT domains could still
function when they were separated from each other (Figure 2).
To characterize the N-terminal TC domains (βγ), two truncated
mutants, PvCPS-N559 (1-559 aa) and PfCPS-N548 (1-548 aa),
were constructed. These mutants exhibited TC activity, but not
PT activity. The PT domains (α) also retained their activity, when
we analyzed the TC domain deletion mutants, PvCPS-C363
(601-963 aa) and PfCPS-C348 (588-935 aa). Therefore, the TC
and PT domains are both able to catalyze their own reactions
without the other domain (Figures S8 to S9). Notably, this
feature is dissimilar from those of other previously reported αβγ
terpene synthases. For example, AgAS, the abietadiene
synthase from Abies grandis (αβγ bifunctional Type I + Type II
TC), shows high interdependence between the two catalytic
domains, because the separate α and βγ domain constructs are
both catalytically inactive.[22] Additionally, in the case of TbTS,
the taxadiene synthase from Taxus brevifolia (αβγ mono
functional Type I TC), both the α and β domains are necessary
for its activity, despite the fact that its active site exists solely in
the α domain.[23] In the case of FCPS/KS, the ent-kaurene
synthase from Phaeosphaeria sp. L487 (αβγ bifunctional Type I
+ Type II TC), although it was possible to construct truncated
mutants that exhibit only one of its two activities,[24] some
interdependence between two catalytic domains still exists,
since the amino acid sequences necessary for the activities of
the N- and C-termini overlap each other. Actually, the region
composed of 172 amino acid residues (359-530 aa) of FCPS/KS,
which contains 946 amino acid residues in total, is required for
both the N- and C-terminus truncated mutants to exhibit their
activities. In contrast, the amino acid sequences of the N- and C-
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
Figure 2. A) GC-MS profiles of products from in vitro enzymatic reactions of
wild-type enzymes, (i) PvCPS, (ii) boiled PvCPS, (iii) PfCPS, (iv) boiled PfCPS,
using DMAPP+IPP as substrates. The chromatograms were extracted at m/z
275 corresponding to [M-Me]+. After the in vitro enzymatic reactions, the
reaction products were dephosphorylated by an acid phosphatase from potato,
and then analyzed by GC-MS. B) and C) The results of in vitro experiments
with wildtype, mutated, and truncated enzymes. B) The results for PvCPS. C)
The results for PfCPS. The experiments with wildtype enzymes indicated that
PvCPS and PfCPS possess both Type II TC and PT activities. The results
from the mutated enzymes suggested that the C-terminal (α) domain and the
N-terminal (βγ) domain are responsible for the PT activity and the Type II TC
activity, respectively. The investigation of truncated enzymes revealed that
both the Type II TC and PT domains are able to exhibit their activities
independently of the other domain.
possess the characteristic linker regions between the α domain
and the βγ domains. There is no such region in the known αβγ
terpene synthases.[1, 3] Thus, not only the catalytic activities but
also the protein architectures substantially differentiate these
new enzymes, PvCPS and PfCPS, from the previously
characterized terpene synthases.
Experimental Section
Strains and Media: Penicillium verruculosum TPU1311 was cultivated at
30 °C, 160 rpm in DPY medium (2% dextrin, 1% hipolypepton (Nihon
Pharmaceutical Co., Ltd.), 0.5% yeast extract (Difco), 0.5% KH2PO4, and
0.05% MgSO4•7H2O) for 2 days, and used as the source for the cloning
of the PvCPS gene. Penicillium fellutanum ATCC 48694 was obtained
from ATCC (Manassas, Virginia). Penicillium fellutanum ATCC 48694
was cultivated at 25 °C, 160 rpm in DPY medium for 3 days, and used as
the source for the cloning of the PfCPS gene. Aspergillus oryzae NSAR1
(niaD-, sC-, ΔargB, adeA-) was used as the host for fungal expression.
Transformants of the A. oryzae strain were grown in shaking cultures in
DPY medium at 30 °C and 160 rpm. The cells were then transferred into
DPY medium with 0.01 % adenine, and the cultures were shaken further
for three or four days. Standard DNA engineering experiments were
performed using Escherichia coli DH5α, purchased from Clontech
(Mountain View, CA). E. coli cells carrying each plasmid were grown in
Luria-Bertani medium and were selected with appropriate antibiotics. E.
coli Rosetta2 (DE3) pLysS (Novagen) was used for the expression of
PvCPS and PfCPS.
The evolution of these unusual αβγ terpene synthases
remains an open question. However, considering the hypothesis
that the architecture of a typical αβγ terpene synthase arose
from the fusion of ancestral genes encoding the discrete α and
βγ proteins,[4] one possibility is that the domain architectures of
PvCPS and PfCPS were also formed by the fusion of separate α
and βγ proteins. The difference is whether the linker region
exists between the two separate catalytic domains.
Notably, such a linker region also exists in αα terpene
synthase.[8, 9] Especially, the comparison with the Type I TC (α) +
PT (α) terpene synthases[8] is significant, because the Type I TC
+ PT terpene synthases and the Type II TC + PT terpene
synthases show high similarity in three ways. 1) Both of them
are TC + PT enzymes, and only the type of the TC is different. 2)
The TC domain exists in the N-terminal region of the enzyme,
while the PT domain is in the C-terminal region, and they are
Secondary structure prediction: For the prediction of secondary
structure in the amino acid sequences of PvCPS and PfCPS, the
SYMPRED server was utilized.[25]
connected by
a linker. 3) In the linker regions, minimal
secondary structure is predicted, according to the amino acid
sequences. These facts suggest that nature adopted the same
strategy with both the Type I TCs and Type II TCs, when
combining the TC with the PT. Importantly, considering that the
evolutionary origins of the Type I TCs and the Type II TCs seem
to be different,[4] they might have independently become fused
with a PT in the course of evolution. Additional studies are
needed to determine the reason why such a highly similar
manner is adopted for both the Type I TCs and Type II TCs.
However, since the fusion of the TC and the PT seems to
accelerate product flux,[10] we believe further investigations of
these enzymes and their homologs will provide valuable insights
into nature’s strategy underlying the evolution of terpene
synthases for the efficient production of terpenoids, which form
one of the largest families of natural products.[1]
Construction of fungal expression plasmids and transformation of
A. oryzae NSAR1: The full-length PvCPS gene was amplified from
Penicillium verruculosum TPU1311 genomic DNA with the primers
pTAex3-PvCPS-F and pTAex3-PvCPS-R (Table S1), using the following
program: 98 °C, 30 s; (98 °C, 10 s; 62 °C, 10 s; 72 °C, 100 s) × 30
cycles; 72 °C, 5 min. The full-length PfCPS gene was amplified from
Penicillium fellutanum ATCC 48694 genomic DNA with the primers
pTAex3-PfCPS-F and pTAex3-PfCPS-R (Table S1), using the following
program: 98 °C, 30 s; (98 °C, 10 s; 61 °C, 10 s; 72 °C, 90 s) × 30 cycles;
72 °C, 5 min. The full-length PvCPS and PfCPS genes were purified, and
ligated into the pTAex3 vector[26], to create pTAex3-PvCPS and pTAex3-
PfCPS, using an In-Fusion® HD Cloning Kit according to the
manufacturer’s protocol. Transformation of A. oryzae NSAR1 was
performed by the protoplast–polyethylene glycol method, as reported
previously.[27]
Gas Chromatographic−Mass Spectrometric Analysis of metabolites
from A. oryzae transformant: For the analysis of the mycelial extract
from the A. oryzae transformant harboring PvCPS or PfCPS, the extract
was subjected to a GC-MS analysis. The temperature of the ionization
chamber was 260 °C, with electron impact ionization at 70 eV. Helium
was used as the carrier gas, and its linear velocity was 44.9 cm/min. The
program held the temperature at 100 °C for 3 min, increased the
temperature at a rate of 14 °C/min up to 330 °C, and then held it at
330 °C for 15 min.
Regarding the genes located nearby the PvCPS and
PfCPS genes, these Type II TC + PT terpene syntheses form
clusters with genes encoding a variety of proteins, including
cytochrome P450s, a short-chain dehydrogenase, an aldehyde
dehydrogenase, and an α/β-hydrolase (Figures S22 to S23). We
are also interested in the functional analyses of these genes.
In conclusion, we identified two unusual αβγ diterpene
synthases and investigated them by in vivo experiments,
heterologous expression in A. oryzae, and in vitro experiments
with wild-type, mutated, and truncated enzymes. As a result,
these enzymes exhibited both Type II TC and PT activities. The
N-terminal (α) domain is responsible for the Type II TC activity to
form 2, and the C-terminal (βγ) domain has the PT activity to
produce GGPP, the substrate for the N-terminal Type II TC
domain. To our knowledge, these are the first terpene synthases
that catalyze both Type II TC and PT reactions. In addition, they
Isolation and purification of 1: For the purification of 1, mycelia from
four liters of the induction culture of the A. oryzae transformant with the
PvCPS gene were extracted with acetone at room temperature overnight.
The extract was concentrated, reextracted with hexane, and then
subjected to silica-gel column chromatography, with elution using a
hexane:ethyl acetate mixture (100:0 to 100:18), to yield 5.7 mg of 1 as a
colorless oil: [α]23 +25.4 (c 0.33, CHCl3); for MS spectrum see Figure
D
S5; for NMR data see Table S2 and Figures S10 to S15; HR-ESI-MS
found m/z 313.2498 [M + Na]+ (calcd. 313.2502 for C20H34NaO). After the
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
cultivation of the A. oryzae transformant with the PfCPS gene in eight
liters of the induction culture, 1 was purified in the same manner, to yield
We thank Prof. K. Gomi (Tohoku University) and Prof. K.
Kitamoto (The University of Tokyo) for the Aspergillus
expression vectors and strain, and Prof. H. Yamazaki (Tohoku
Pharmaceutical University) for the P. verruculosum strain. This
work was supported in part by a Grant-in-Aid for Scientific
Research from the MEXT, Japan (JSPS KAKENHI Grant
Number JP15H01836, JP16H06443, JP26303005, JP24688011,
and JP17H05429), and JSPS Research Fellowships for Young
Scientists (to T.M.).
55.6 mg of 1 as a colorless oil: [α]31 +22.3 (c 1.00, CHCl3); for MS
spectrum see Figure S5; for NMR data see Table S3 and Figures S16 to
S21; HR-ESI-MS found m/z 313.2508 [M + Na]+ (calcd. 313.2502 for
C20H34NaO).
D
Expression and purification of Recombinant Proteins: The intron-free
PvCPS gene was amplified with the primers pColdTF-PvCPS-F and
pColdTF-PvCPS-R (Table S1), from the cDNA synthesized from RNA
extracted from the A. oryzae transformant with the PvCPS gene, using
the following program: 98 °C, 30 s; (98 °C, 10 s; 62 °C, 10 s; 72 °C, 90 s)
× 30 cycles; 72 °C, 5 min. To obtain the intron-free PfCPS gene, the
exons were amplified with the primers pColdTF-PfCPS-F1/ pColdTF-
PfCPS-R1 and pColdTF-PfCPS-F2/ pColdTF-PfCPS-R2 (Table S1) from
the pTAex3-PfCPS, using the following program: 98 °C, 30 s; (98 °C, 10
s; 61 °C, 10 s; 72 °C, 30 s) × 30 cycles; 72 °C, 5 min. After the
amplification, the PvCPS cDNA and the PfCPS cDNA fragments were
purified, and ligated into the pColdTF vector (TaKaRa), to create
pColdTF-PvCPS and pColdTF-PfCPS, using an In-Fusion® HD Cloning
Kit according to the manufacturer’s protocol. The full-length plasmids for
the production of mutated or truncated PvCPS and PfCPS were amplified
with the appropriate primers (Table S1) from pColdTF-PvCPS and
pColdTF-PfCPS, using the following program: 98 °C, 30 s; (98 °C, 10 s;
65 °C, 10 s; 72 °C, 128 s) × 30 cycles; 72 °C, 5 min, as the linear form.
After the amplification, the fragments were purified, treated with DpnI
(TAKARA), and converted into the circular form, using T4 Polynucleotide
Kinase (TOYOBO) and a DNA Ligation Kit Ver. 2.1 (TAKARA). E. coli
Rosetta2 (DE3) pLysS (Novagen) cells were transformed with each
plasmid. E. coli Rosetta 2 (DE3) pLysS transformants were cultured to an
OD600 of 0.5 in LB medium containing 100 mg/L ampicillin at 37 °C.
Keywords: biosynthesis • terpenoids • bifunctional enzyme •
terpene cyclase • prenyltransferase
[1]
a) D. W. Christianson, Chem. Rev. 2006, 106, 3412-3442; b) D. W.
Christianson, Curr. Opin. Chem. Biol. 2008, 12, 141-150.
[2]
[3]
C. D. Poulter, H. C. Rilling, Acc. Chem. Res. 1978, 11, 307-313.
Y. Gao, R. B. Honzatko, R. J. Peters, Nat. Prod. Rep. 2012, 29, 1153-
1175.
[4]
a) R. Cao, Y. Zhang, F. M. Mann, C. Huang, D. Mukkamala, M. P.
Hudock, M. E. Mead, S. Prisic, K. Wang, F. Y. Lin, T. K. Chang, R. J.
Peters, E. Oldfield, Proteins: Struct., Funct., Bioinf. 2010, 78, 2417-
2432; b) E. Oldfield, F. Y. Lin, Angew. Chem., Int. Ed. 2012, 51, 1124-
1137.
[5]
[6]
D. Reardon, G. K. Farber, FASEB J. 1995, 9, 497-503.
M. Chen, G. G. Harris, T. A. Pemberton, D. W. Christianson, Curr. Opin.
Struct. Biol. 2016, 41, 27-37.
[7]
[8]
a) T. Toyomasu, Biosci., Biotechnol., Biochem. 2008, 72, 1168-1175; b)
H. Y. Gong, Y. Zeng, X. Y. Chen, Nat. Prod. Bioprospect. 2014, 4, 59-
72.
Isopropyl β-D-1-thiogalactopyranoside was then added to
a final
a) Y. Matsuda, T. Mitsuhashi, Z. Quan, I. Abe, Org. Lett. 2015, 17,
4644-4647; b) Y. Matsuda, T. Mitsuhashi, S. Lee, M. Hoshino, T. Mori,
M. Okada, H. Zhang, F. Hayashi, M. Fujita, I. Abe, Angew. Chem., Int.
Ed. 2016, 55, 5785-5788; c) B. Qin, Y. Matsuda, T. Mori, M. Okada, Z.
Quan, T. Mitsuhashi, T. Wakimoto, I. Abe, Angew. Chem., Int. Ed. 2016,
55, 1658-1661; d) M. Okada, Y. Matsuda, T. Mitsuhashi, S. Hoshino, T.
Mori, K. Nakagawa, Z. Quan, B. Qin, H. Zhang, F. Hayashi, H. Kawaide,
I. Abe, J. Am. Chem. Soc. 2016, 138, 10011-10018; e) T. Mitsuhashi, J.
Rinkel, M. Okada, I. Abe, J. S. Dickschat, Chem. - Eur. J. 2017; f) R.
Chiba, A. Minami, K. Gomi, H. Oikawa, Org. Lett. 2013, 15, 594-597; g)
Y. Ye, A. Minami, A. Mandi, C. Liu, T. Taniguchi, T. Kuzuyama, K.
Monde, K. Gomi, H. Oikawa, J. Am. Chem. Soc. 2015, 137, 11846-
11853; h) T. Toyomasu, M. Tsukahara, A. Kaneko, R. Niida, W.
Mitsuhashi, T. Dairi, N. Kato, T. Sassa, Proc. Natl. Acad. Sci. U. S. A.
2007, 104, 3084-3088; i) T. Toyomasu, A. Kaneko, T. Tokiwano, Y.
Kanno, Y. Kanno, R. Niida, S. Miura, T. Nishioka, C. Ikeda, W.
Mitsuhashi, T. Dairi, T. Kawano, H. Oikawa, N. Kato, T. Sassa, J. Org.
Chem. 2009, 74, 1541-1548; j) G. Bian, Y. Han, A. Hou, Y. Yuan, X. Liu,
Z. Deng, T. Liu, Metab. Eng. 2017, 42, 1-8.; k) A. Minami, N. Tajima, Y.
Higuchi, T. Toyomasu, T. Sassa, N. Kato, T. Dairi, Bioorg. Med. Chem.
Lett. 2009, 19, 870-874.
concentration of 0.50 mM to induce gene expression, and the cultures
were incubated further for 18 h at 15 °C. E. coli cells were harvested by
centrifugation at 5,800 x g and resuspended in 50 mM Tris-HCl buffer
(pH 8.4), containing 250 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl2, and
5 mM imidazole (buffer A). The cells were disrupted by sonication, and
the lysate was centrifuged at 7,300 x g for 20 min. The supernatant was
loaded onto
a
Cosmogel His-Accept (Nacalai Tesque) column
equilibrated with buffer A. After the resin was washed with buffer A
containing 10 mM imidazole, the recombinant proteins were
subsequently eluted with buffer A containing 300 mM imidazole. The
protein purification buffer was replaced with 50 mM Tris-HCl buffer (pH
8.4), containing 250 mM NaCl, 10% (v/v) glycerol, and 5 mM MgCl2. All
purification procedures were performed at 4 °C.
Reaction of Recombinant Proteins: The standard enzymatic reaction
was performed in mixtures containing 100 mM Tris-HCl (pH 7.5),
substrates (10 μg of DMAPP + 10 μg of IPP, 10 μg of GPP + 10 μg of
IPP, 10 μg of FPP + 10 μg of IPP, 3 μg of GGPP or 10 μg of GFPP), 5
mM MgCl2, 2 mM dithiothreitol, and 4.7 mM enzyme, in a final volume of
100 μL. When GGPP was utilized as the substrate, the final volume was
300 μL. In experiments with mutated or truncated enzymes, GGPP was
used as the substrate to monitor the TC activity. When the PT activity
was investigated, we used FPP+IPP as substrates. After an incubation at
30 °C overnight, the enzyme product was extracted with water-saturated
[9]
a) G. G. Harris, P. M. Lombardi, T. A. Pemberton, T. Matsui, T. M.
Weiss, K. E. Cole, M. Köksal, F. V. Murphy IV, L. S. Vedula, W. K. W.
Chou, D. E. Cane, D. W. Christianson, Biochemistry 2015, 54, 7142-
7155; b) J. Jiang, X. He, D. E. Cane, Nat. Chem. Biol. 2007, 3, 711-
715; c) J. Jiang, X. He, D. E. Cane, J. Am. Chem. Soc. 2006, 128,
8128-8129; d) D. E. Cane, R. M. Watt, Proc. Natl. Acad. Sci. U. S. A.
2003, 100, 1547-1551; e) B. Gust, G. L. Challis, K. Fowler, T. Kieser, K.
F. Chater, Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 1541-1546.
butanol, and then the butanol was removed by evaporation.
A
dephosphorylation reaction was then performed in mixtures containing
20% (v/v) 1-propanol, 100 mM sodium acetate (pH 5.0), 0.1% (v/v) Triton
X-100, and 0.13 units of acid phosphatase from potato (Sigma-Aldrich),
in a final volume of 500 μL. After an incubation at 37 °C overnight and an
extraction with hexane, the enzyme products were analyzed by GC-MS.
The GC-MS conditions were the same as those for the analysis of the
mycelial extracts from the A. oryzae transformants harboring PvCPS or
PfCPS.
[10] a) P. Bauler, G. Huber, T. Leyh, J. A. McCammon, J. Phys. Chem. Lett.
2010, 1, 1332-1335; b) M. Brodelius, A. Lundgren, P. Mercke, P. E.
Brodelius, Eur. J. Biochem. 2002, 269, 3570-3577.
[11] M. Castellana, M. Z. Wilson, Y. Xu, P. Joshi, I. M. Cristea, J. D.
Rabinowitz, Z. Gitai, N. S. Wingreen, Nat. Biotechnol. 2014, 32, 1011-
1018.
[12] M. Chen, W. K. W. Chou, T. Toyomasu, D. E. Cane, D. W. Christianson,
ACS Chem. Biol. 2016, 11, 889-899.
Acknowledgements
[13] H. Yamazaki, W. Nakayama, O. Takahashi, R. Kirikoshi, Y. Izumikawa,
K. Iwasaki, K. Toraiwa, K. Ukai, H. Rotinsulu, D. S. Wewengkang, D. A.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
Sumilat, R. E. P. Mangindaan, M. Namikoshi, Bioorg. Med. Chem. Lett.
2015, 25, 3087-3090.ꢀ
[14] M. J. Smanski, Z. Yu, J. Casper, S. Lin, R. M. Peterson, Y. Chen, E.
Wendt-Pienkowski, S. R. Rajski, B. Shen, Proc. Natl. Acad. Sci. U. S. A.
2011, 108, 13498-13503.
[15] J. D. Rudolf, L. B. Dong, H. Cao, C. Hatzos-Skintges, J. Osipiuk, M.
Endres, C. Y. Chang, M. Ma, G. Babnigg, A. Joachimiak, G. N. Phillips,
Jr., B. Shen, J. Am. Chem. Soc. 2016, 138, 10905-10915.
[16] a) M. Köksal, Y. Jin, R. M. Coates, R. Croteau, D. W. Christianson,
Nature 2011, 469, 116-120; b) K. Zhou, Y. Gao, J. A. Hoy, F. M. Mann,
R. B. Honzatko, R. J. Peters, J. Biol. Chem. 2012, 287, 6840-6850; c)
M. Köksal, H. Hu, R. M. Coates, R. J. Peters, D. W. Christianson, Nat.
Chem. Biol. 2011, 7, 431-433.
[17] F. J. Jin, J. Maruyama, P. R. Juvvadi, M. Arioka, K. Kitamoto, FEMS
Microbiol. Lett. 2004, 239, 79-85.
[18] a) Y. Matsuda, T. Iwabuchi, T. Wakimoto, T. Awakawa, I. Abe, J. Am.
Chem. Soc. 2015, 137, 3393-3401; b) Y. Matsuda, T. Wakimoto, T.
Mori, T. Awakawa, I. Abe, J. Am. Chem. Soc. 2014, 136, 15326-15336.
[19] H. Toshima, H. Oikawa, T. Toyomasu, T. Sassa, Tetrahedron 2000, 56,
8443-8450.
[20] Y. Wu, K. Zhou, T. Toyomasu, C. Sugawara, M. Oku, S. Abe, M. Usui,
W. Mitsuhashi, M. Chono, P. M. Chandler, R. J. Peters, Phytochemistry
2012, 84, 40-46.
[21] a) S. Prisic, J. Xu, R. M. Coates, R. J. Peters, ChemBioChem 2007, 8,
869-874; b) A. Chen, C. D. Poulter, P. A. Kroon, Protein Sci. 1994, 3,
600-607.
[22] R. J. Peters, O. A. Carter, Y. Zhang, B. W. Matthews, R. B. Croteau,
Biochemistry 2003, 42, 2700-2707.
[23] T. A. Pemberton, M. Chen, G. G. Harris, W. K. W. Chou, L. Duan, M.
Köksal, A. S. Genshaft, D. E. Cane, D. W. Christianson, Biochemistry
2017, 56, 2010-2023.
[24] H. Kawaide, T. Sassa, Y. Kamiya, J. Biol. Chem. 2000, 275, 2276-2280.
[25] V. A. Simossis, J. Heringa, Comput. Biol. Chem. 2004, 28, 351-366.
[26] T. Fujii, H. Yamaoka, K. Gomi, K. Kitamoto, C. Kumaga, Biosci.,
Biotechnol., Biochem. 1995, 59, 1869-1874.
[27] K. Gomi, Y. Iimura, S. Hara, Agric. Biol. Chem. 1987, 51, 2549-2555.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201700445
ChemBioChem
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
We identified two unusual diterpene
synthases composed of three
Takaaki Mitsuhashi, Masahiro Okada,
and Ikuro Abe*
domains (α, β, and γ) and one
Page No. – Page No.
characteristic linker inserted between
α domain and βγ domain. They are
the first enzymes possessing both
Type II terpene cyclase (TC) and
prenyltransferase (PT) activities.
Identification of Chimeric αβγ
Diterpene Synthases Possessing
both Type II Terpene Cyclase and
Prenyltransferase Activities
Layout 2:
COMMUNICATION
Author(s), Corresponding Author(s)*
((Insert TOC Graphic here))
Page No. – Page No.
Title
Text for Table of Contents
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.