4-O-acetylation and 3-O-acetylation of trichothecenes by trichothecene 15-O-acetyltransferase encoded by Fusarium Tri3

Article abstract of DOI:10.1271/bbb.80501

In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50μM), we observed 3-O-acetylation activity against isotrichodermol that was more than 105 times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15- diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.

Full text of DOI:10.1271/bbb.80501

Biosci. Biotechnol. Biochem., 72 (9), 2485–2489, 2008
Communication
4-O-Acetylation and 3-O-Acetylation of Trichothecenes
by Trichothecene 15-O-Acetyltransferase Encoded by Fusarium Tri3
1;2
Takeshi TOKAI,1;
Naoko TAKAHASHI-ANDO,
Masumi IZAWA,
*
;***
1;**,***
y
1;3;
Takashi KAMAKURA,2 Minoru YOSHIDA,3 Makoto FUJIMURA,4 and Makoto KIMURA
1Plant & Microbial Metabolic Engineering Research Unit, Discovery Research Institute (DRI), RIKEN,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2Faculty of Science and Technology, Tokyo University of Science,
2461 Yamazaki, Noda, Chiba 278-8510, Japan
3Chemical Genetics Laboratory, Advanced Science Institute (ASI), RIKEN,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
4Faculty of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura, Gunma 374-0193, Japan
Received July 22, 2008; Accepted August 12, 2008; Online Publication, September 7, 2008
[doi:10.1271/bbb.80501]
In the biosynthesis of Fusarium trichothecenes, the
C-3 hydroxyl group of isotrichodermol must be acety-
lated by TRI101 for subsequent pathway genes to
function. Despite the importance of this 3-O-acetylation
step in biosynthesis, Tri101 is both physically and
evolutionarily unrelated to other Tri genes in the
trichothecene gene cluster. To gain insight into the
evolutionary history of the cluster, we purified recombi-
nant TRI3 (rTRI3), one of the two cluster gene-encoded
trichothecene O-acetyltransferases, and examined to
determine whether this 15-O-acetyltransferase can add
an acetyl to the C-3 hydroxyl group of isotrichodermol.
When a high concentration of rTRI3 was used in the
assay (final concentration, 50 ꢀM), we observed 3-O-
acetylation activity against isotrichodermol that was
more than 105 times less efficient than the known 15-O-
acetylation activity against 15-deacetylcalonectrin. The
rTRI3 protein also exhibited 4-O-acetylation activity
when nivalenol was used as a substrate; in addition to
15-acetylnivalenol, di-acetylated derivatives, 4,15-diace-
tylnivalenol, and, to a lesser extent, 3,15-diacetylniva-
lenol, were also detected at high enzyme concentrations.
The significance of the trace trichothecene 3-O-acetyl-
transferase activity detected in rTRI3 is discussed in
relation to the evolution of the trichothecene gene
cluster.
Key words: biosynthesis gene cluster; fungal secondary
metabolism; Fusarium graminearum (Gib-
berella zeae); sesquiterpene; trichothecene
mycotoxins
Fungal genes involved in secondary metabolism
often compose a unit with all the genes necessary for
production of the metabolite clustered together in the
host genome. To explain this self-contained nature of
secondary metabolite gene clusters, Walton hypothe-
sized that a selective advantage is conferred on the
genes that comprise the cluster by the clustering,
separately from any advantage that the product of that
cluster confers on the host organism.1) Occasionally,
the secondary metabolite genes are duplicated, sepa-
rated, and/or only partially clustered due to known
genomic forces, such as translocation, inversion, and
unequal crossing-over. Examples include the genes for
HC-toxin biosynthesis in Cochliobolus carbonum2) and
the genes for T-toxin biosynthesis in Cochliobolus
heterostrophus.3) Nevertheless, all the genes necessary
for toxin production are thought to have occurred
originally in a single gene cluster in either case. Hence,
the ancestral secondary metabolite gene clusters are
thought to have been self-contained (complete) when
the producing fungi acquired them by horizontal gene
transfer.
y
To whom correspondence should be addressed. Fax: +81-48-462-4394; E-mail: mkimura@riken.jp
Present address: Department of Agriculture, Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa 243-0034, Japan
Present address: Department of Applied Chemistry, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan
TT initially made all the new findings presented in this paper. NTA re-confirmed the findings by further analysis and obtained the main data for
presentation.
*
**
***
Abbreviations: 3-ADON, 3-acetyldeoxynivalenol; 15-ADON, 15-acetyldeoxynivalenol; 3-ANIV, 3-acetylnivalenol; 4-ANIV, 4-acetylnivalenol;
15-ANIV, 15-acetylnivalenol; CAL, calonectrin; 15-deCAL, 15-deacetylcalonectrin; 3,4-diANIV, 3,4-diacetylnivalenol; 3,15-diADON, 3,15-
diacetyldeoxynivalenol; 3,15-diANIV, 3,15-diacetylnivalenol; 4,15-diANIV, 4,15-diacetylnivalenol; DON, deoxynivalenol; ITD, isotrichodermin;
NIV, nivalenol; 3,4,15-triANIV, 3,4,15-triacetylnivalenol

2486
T. TOKAI et al.
In the case of trichothecene biosynthesis in Fusarium
and pI have been calculated to be 57,878 and 5.12
respectively.
species, the extant trichothecene gene cluster is not self-
contained.4,5) Most of the pathway genes cannot function
unless the C-3 hydroxyl group of isotrichodermol, the
first pathway intermediate with a toxic trichothecene
skeleton, is acetylated by a pathway enzyme encoded
by Tri101.6,7) This gene is a non-cluster trichothecene
gene with a different evolutionary history from other
Tri genes;8,9) that is, the possibility that Tri101 was
originally in the gene cluster and later separated from
other Tri genes was unambiguously excluded.8,10,11) Its
functional homolog has also been found in several other
ascomycetes, but mostly with much less catalytic
activity than the trichothecene-producer TRI101.11)
Therefore, only few fungal species appear to serve as
suitable hosts for the persistence of the non-self-con-
tained (incomplete) trichothecene gene cluster. Given
the importance of complete clustering to the persistence
of secondary metabolite genes,1) it might be necessary
to consider a scenario alternative to the fortuitous
acquisition and propagation of a Tri101-dependent
gene cluster.
To determine whether traces of trichothecene 3-O-
acetyltransferase activity exist among known acetylases
encoded by the trichothecene cluster genes, we have
undertaken expression of Tri3 (known as the trichothe-
cene 15-O-acetyltransferase gene responsible for acety-
lation at C-15 of 15-deacetylcalonectrin, 15-deCAL, in
the biosynthesis of trichothecenes)12) and Tri7 (known as
the trichothecene 4-O-acetyltransferase gene)13) in Esch-
erichia coli. Fusarium graminearum MAFF 111233
(F. graminearum lineage 6, newly named Fusarium
asiaticum)14) was used as the source of trichothecene
biosynthetic genes in this study. In the construction of
the expression vector, cDNA was synthesized from
RNA isolated from mycelia grown in RF medium15)
using a Superscript IIIꢀ First-Strand Synthesis system
(Invitrogen, Carlsbad, CA). Among the two acetyltrans-
ferases, Tri7 failed to be expressed at a detectable level
by any means. Hence, we focused on the purification
and characterization of recombinant TRI3, whose cDNA
was successfully expressed at a high level in E. coli,
as follows: (i) the entire coding region of Tri3 was
amplified by PCR with primers MFTri3ATG/Nde
(50-TCATATGAGCGCTTCATCCTCCGCCTTG-30) and
MFTri3TAA/Bam (50-TGGATCCTTACAATTTGAA-
TGCCAGCATGA-30) using KOD-plus DNA poly-
merase (Toyobo, Osaka, Japan), (ii) the NdeI-BamHI
fragment of the amplified Tri3 product was inserted
between the corresponding sites of a pCold IIIꢀ
expression vector (Takara Bio, Kusatsu, Japan), and
(iii) the resulting vector, pColdIII-MFTri3, was trans-
formed to E. coli Rosettaꢀ 2 (DE3) competent cells
(Novagen, Madison, WI) for production of the recombi-
nant enzyme. The recombinant TRI3 protein produced
from this vector (hereafter referred to as rTRI3) contains
N-terminal extensions of six amino acid residues
derived from the pCold IIIꢀ vector. Its theoretical Mr
After overexpression of Tri3 following the protocol
of the cold shock expression system (Takara Bio), a
recombinant protein fraction was prepared from dis-
rupted bacterial cells as described previously.16) The
sample was then applied to a HiTrap S HP (5 ml)
column (GE Healthcare UK, Buckinghamshire, UK)
equilibrated with buffer A (20 mM Tris–HCl, pH 6.5).
While most proteins were trapped to the cation exchange
column at this pH, rTRI3 readily passed through the
column. The flow-through fraction was concentrated
with Centriprep-30/Centricon-30 (Millipore, Billerica,
MA) and further purified by gel permeation chromatog-
raphy with a Superdex 75 HR10/30 column (GE
Healthcare UK) using buffer A containing 0.1 M NaCl.
The rTRI3 protein was eluted as a single band at about
9.6 ml (Mr 55 k) (Supplemental Fig. 1A; see Biosci.
Biotechnol. Biochem. Web site). The enzyme concen-
tration was determined using the BCA protein assay kit
(Thermo Fisher Scientific, Rockford, IL). When deoxy-
nivalenol (DON) was used as the substrate, the purified
enzyme showed maximal C-15 acetylation activity at
pH 6.5–7.5 (Supplemental Fig. 1B), and at 15–37 ꢀC
(Supplemental Fig. 1C).
On HPLC analysis, it is not possible to monitor the
trichothecene pathway’s intermediates (which lack a
keto at C-8) by measuring UV absorbance at 254 nm.
Hence, TLC was used to estimate roughly the amount of
the acetylated trichothecene product. Samples were
developed on a TLC plate using ethyl acetate/toluene
(3:1) as the solvent, and trichothecenes separated on the
plate were visualized by a color reaction using a chro-
mogenic reagent, 4-(p-nitrobenzyl)pyridine (NBP).17)
Substrates trichothecene and acetyl-CoA (final con-
centrations, 0.1 mg/ml and 2 mM respectively) were
mixed with various concentrations of rTRI3 in 100 mM
Tris–HCl, pH 7.0, and the reaction was carried out at
30 ꢀC. The enzyme concentration and incubation time
were adjusted so that the reaction mixture contained
only similar low levels of acetylated product on a TLC
plate.
When the assay was carried out using a high
concentration of rTRI3 protein (50 mM), acetylation of
a small fraction of isotrichodermol giving isotrichoder-
min (ITD) was observed with 18 h of incubation (see
Fig. 1A). This result indicates that TRI3 has trace
activity against the C-3 hydroxyl group of isotrichoder-
mol. To control the acetylation reaction to a level as low
as that obtained as above using the regular substrate 15-
deCAL, the enzyme concentration and incubation time
had to be reduced to 5 nM (1=104 concentration) and 1 h
(1/18 time) (Fig. 1B). This means that the 3-O-acety-
lation activity against isotrichodermol was lower than
the 15-O-acetylation activity against 15-deCAL on the
order of 105 or more. When 3-acetyldeoxynivalenol
(3-ADON) and DON were used as substrate and the
reaction was allowed to proceed for 1 h, the amount of

Characterization of TRI3 Acetylase
2487
A
B
Substrate
rTRI3
Substrate
rTRI3
Substrate
isotrichodermol
15-deCAL
5 nM
3-ADON DON
50 nM 1 µM
rTRI3
50 µM
O
OAc
OAc
OH
O
O
O
O
O
OAc
OH
O
OAc
3,15-diADON
AcO
O
OAc
CAL
O
O
O
O
OAc
O
OH
OH
ITD
O
3-ADON
HO
15-deCAL
O
OH
O
O
OH
OAc
isotrichodermol
15-ADON
O
OH
O
O
OH
OH
DON
6 h 18 h
1 h 2 h
1 h 2 h 1 h 2 h
Fig. 1. The TRI3 Enzyme Exhibits Trace Acetylation Activity against the C-3 Hydroxyl Group of Isotrichodemol.
After the enzyme reaction, trichothecenes were extracted with ethyl acetate, and aliquots (approximately 25 mg) were separated on TLC plates.
Substrate trichothecenes and enzyme concentrations are indicated at the top, and incubation times are indicated at the bottom. Trichothecene
standards loaded on the TLC plates are indicated by arrows. A, 3-O-Acetylation of isotrichodermol by rTRI3. Isotrichodermol was purified from
the Tri101ꢁ mutant of F. graminearum.20) Formation of ITD was also confirmed by GC-MS analysis (data not shown). B, 15-O-Acetylation of
DON-type trichothecenes by rTRI3. Three known substrates, 15-deCAL, 3-ADON, and DON, were used in the acetylation assay to roughly
evaluate the 3-O-acetylation activity against isotrichodermol. 15-deCAL, calonectrin (CAL), 3,15-diacetyldeoxynivalenol (3,15-diADON), and
15-acetyldeoxynivalenol (15-ADON) were purified from the Triꢁ mutants of F. graminearum,20) and 3-ADON and DON were purchased from
Wako Pure Chemical Industries (Osaka, Japan).
the enzyme necessary to achieve similar levels of 15-O-
acetylation increased on the order of 10 (50 nM) and 200
(1 mM) respectively, as compared to that obtained with
15-deCAL (Fig. 1B). Nevertheless, the 15-O-acetylation
activities against these type B trichothecenes were still
much greater than the 3-O-acetylation activity against
isotrichodermol. The extremely weak activity of TRI3
against isotrichodermol does not contradict a previous
report that Tri101ꢁ mutants (carrying a functional copy
of Tri3) of Fusarium sporotrichioides were blocked in
their ability to produce T-2 toxin and accumulated
isotrichodermol.7)
To examine the enzymatic properties of TRI3 in more
detail, we used nivalenol (NIV) next as a substrate. The
reaction was carried out for 12 h with different concen-
trations of the enzyme. As shown in Fig. 2A, the C-15
hydroxyl group of NIV was first acetylated to give 15-
acetynivalenol (15-ANIV). Completion of C-15 acety-
lation was then followed by C-4 acetylation or, to a
lesser extent, C-3 acetylation, to give 4,15-diacetylni-
valenol (4,15-diANIV) and 3,15-diacetylnivalenol (3,15-
diANIV) respectively, albeit with much less efficiency
than C-15 acetylation. This result indicates that TRI3
is a multifunctional enzyme that can acetylate two
of the three hydroxyl groups, the C-15 and C-4 hydroxyl
groups or, to a lesser extent, the C-15 and C-3 hydroxyl
groups, attached to the trichothecene skeleton. The
acetylation of two hydroxyl groups is similar to the
action of chloramphenicol acetyltransferase (CAT),
which acetylates the antibiotic at one (chloramphenicol
3-acetate) or both (chloramphenicol 1,3-diacetate) of its
two hydroxyl groups. However, CAT can add an acetyl
only to the first acetylation site of chloramphenicol,
and the second acetylation is based on re-acetylation
of the first site during the non-enzymatic intercon-
version of mono-acetylated derivatives at higher pH
values.18) In this respect, TRI3 is a novel type of multi-
functional acetylase distinct from CAT and its related
acetylases, although it belongs to the CAT class of
acetyltransferases containing the consensus sequence
HXXXDG.19)
We also used all possible mono- and di-acetylated
NIV derivatives as substrate in subsequent analyses: 3-
acetylnivalenol (3-ANIV), 4-acetylnivalenol (4-ANIV),
15-ANIV, 3,4-diacetylnivalenol (3,4-diANIV), 3,15-
diANIV, and 4,15-diANIV (see Fig. 2B). Based on the
enzyme concentration and incubation time used in the
assay, the following properties of TRI3 were identified:
(i) the presence of an acetyl at C-3 significantly
accelerates 15-O-acetylation (compare lane 3-ANIV
and Fig. 2A), (ii) the presence of an acetyl at C-4
blocks 15-O-acetylation (see lane 4-ANIV), (iii) the
presence of both C-3 and C-4 acetyls allows an
extremely limited level of 15-O-acetylation (see lane
3,4-diANIV), and (iv) neither C-4 nor C-3 can be
acetylated if the other two positions are occupied by
acetyls (see lanes 3,15-diANIV and 4,15-diANIV).
Considering that the biosynthetic pathway of NIV-type
trichothecenes 4,15-diANIV/4-ANIV proceed via 3,15-
diANIV and 3,4,15-triacetylnivalenol (3,4,15-triANIV),

2488
T. TOKAI et al.
A
B
50 µM
rTRI3
Substrate
rTRI3
NIV
Substrate
rTRI3
10 nM 1 µM 5 µM 20 µM
100 nM 2 µM 10 µM 50 µM
Substrate
50 nM
500 nM
O
OAc
OAc
O
3,4,15-triANIV
O
OH
O
OAc
O
O
OAc
3,4-diANIV
OAc
OH
3,15-diANIV
3,15-diANIV
O
O
O
O
OAc
OH
OH
OH
OH
OAc
O
4,15-diANIV
OH
4,15-diANIV
O
OH
O
OAc
O
4-ANIV
OAc
O
OAc
OH
O
OH
OAc
O
3-ANIV
OH
O
O
OH
OH
15-ANIV
OH
OH
O
15-ANIV
O
OH
OAc
12 h
1 h 2 h
12 h
Fig. 2. The TRI3 Enzyme Acetylates the Hydroxyl Groups at C-15, C-4, and/or C-3 of NIV-Type Trichothecenes.
After the enzyme reaction, trichothecenes were extracted with ethyl acetate, and aliquots (approximately 25 mg) were separated on TLC plates.
Substrate trichothecenes and enzyme concentrations are indicated at the top, and incubation times are indicated at the bottom. Trichothecene
standards loaded on the TLC plates are indicated by arrows. A, Acetylation of NIV by rTRI3. The substrate NIV (Wako) was hardly detected on
the TLC plate, because only a limited amount of this mycotoxin is extractable with ethyl acetate. The reaction products were also analyzed by
GC-MS to confirm their structures (data not shown). B, Acetylation of mono- and di-acetylated NIV derivatives by rTRI3. A small amount of 3-
ANIV was produced when 3,15-diANIV was used as the substrate. This reverse reaction is catalyzed by the TRI3 enzyme and not by an
endogenous deacetylase derived from E. coli, because no such product was detected in the control reaction mixture (data not shown). 4-ANIV
(fusarenone X) was purchased from Wako. 3-ANIV and 3,4-diANIV were prepared by in vitro acetylation of NIV and 4-ANIV respectively by
TRI101.6) 4,15-diANIV was purified from wild-type F. graminearum MAFF 111233, and 15-ANIV and 3,15-diANIV were purified from the
Tri7ꢁ mutant.20)
in that order,20) the inability of TRI3 to acetylate the
three positions at the same time, as indicated in (iv),
necessitated the involvement of Tri7 at the last acety-
lation step at C-4. This is in consistent with the results of
previous targeted gene-disruption experiments, in which
deletion of Tri7 (with a functional copy of Tri3) caused
the C-4 hydroxyl group of trichothecenes no longer to be
acetylated.13,21)
In view of the highly unstable nature of fungal
genomes,22) secondary metabolite pathway genes in
complete gene clusters have greater chances of persis-
tence than dispersed pathway genes, because the former
can be transmitted horizontally: if genes for a novel
metabolic process can move by horizontal gene trans-
fer, then they have a reasonable chance of conferring
a new selective advantage on the new host, which
promotes the survival of the new host and also of the
genes.1) In addition to these advantages of clustering,
secondary metabolite gene clusters might be maintained
by the advantage that they are subject to efficient
transcriptional regulation, presumably at the level of
chromatin structure.23) To date, the only scenario for
the evolution of the trichothecene gene cluster has been
that an ancestral Fusarium species, possessing a strong
trichothecene 3-O-acetyltransferase activity for tricho-
thecene resistance, fortuitously acquired the non-self-
contained trichothecene gene cluster. However, secon-
dary metabolite genes in an incomplete gene cluster
are evolutionarily less advantageous for persistence
than those in a complete gene cluster.1) An alternative
scenario is that the ancestral trichothecene gene cluster
originally contained all the genes necessary for the
biosynthesis of its product. Although the contribution of
Tri3 in extant trichothecene-producing Fusarium is too
small to feature in the biosynthetic step carried out by
Tri101 (isotrichodermol ! ITD), the trace 3-O-acety-
lation activity discovered in this study suggests the self-
contained nature of the ancestral trichothecene gene
cluster. If this is the case, the presence of two
functional enzymes for 3-O-acetylation (TRI101 and
TRI3) led to a relaxation of selective constraints, which
eventually resulted in the loss of the trichothecene
3-O-acetyltransferase activity of TRI3. In either case,
the 3-O-acetylation step and its enzymes are unique
and important in the biosynthesis of Fusarium tricho-
thecenes.
Acknowledgments
This research was supported by a Grant-in-Aid for
Scientific Research (C) (grant 20580117) from the Japan
Society for the Promotion of Science (JSPS).

Characterization of TRI3 Acetylase
2489
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