Physically Discrete β-Lactamase-Type Thioesterase Catalyzes Product Release in Atrochrysone Synthesis by Iterative Type I Polyketide Synthase

Article abstract of DOI:10.1016/j.chembiol.2009.04.004

ATEG_08451 in Aspergillus terreus, here named atrochrysone carboxylic acid synthase (ACAS), is a nonreducing, iterative type I polyketide synthase that contains no thioesterase domain. In vitro, reactions of ACAS with malonyl-CoA yielded a polyketide inte

Full text of DOI:10.1016/j.chembiol.2009.04.004

Chemistry & Biology
Article
Physically Discrete b-Lactamase-Type Thioesterase
Catalyzes Product Release in Atrochrysone
Synthesis by Iterative Type I Polyketide Synthase
Takayoshi Awakawa,1 Kosuke Yokota,1 Nobutaka Funa,1 Fuminao Doi,2 Naoki Mori,2 Hidenori Watanabe,2
and Sueharu Horinouchi1,
*
1Department of Biotechnology
2Department of Applied Biological Chemistry
Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
*Correspondence: asuhori@mail.ecc.u-tokyo.ac.jp
DOI 10.1016/j.chembiol.2009.04.004
SUMMARY
formation of a new carbon-carbon bond. The decarboxylative
Claisen condensation occurs iteratively, catalyzed by a single
ATEG_08451 in Aspergillus terreus, here named module, and the polyketide chain is thus extended. Additionally,
b-ketoreductase (KR), dehydratase (DH), and enoyl reductase
atrochrysone carboxylic acid synthase (ACAS), is
(ER) domains are sometimes included in the system to synthe-
size reduced compounds.
Among the iterative type I PKSs, those consisting of KS, AT,
a nonreducing, iterative type I polyketide synthase
that contains no thioesterase domain. In vitro, reac-
tions of ACAS with malonyl-CoA yielded a polyketide
intermediate, probably attached to its acyl carrier
protein (ACP). The addition of ATEG_08450, here
named atrochrysone carboxyl ACP thioesterase
and ACP domains and possessing no reducing domains are
classified as nonreducing PKSs (NR-PKSs). In addition to KS,
AT, and ACP domains, NR-PKSs have some unique domains,
such as a starter unit ACP transacylase (SAT; Crawford et al.,
(ACTE), to the reaction resulted in the release of
products derived from atrochrysone carboxylic
acid, such as atrochrysone and endocrocin. ACTE,
2006), product template (PT; Crawford et al., 2008), and thioes-
terase (TE) domains (Fujii et al., 2001). A general pattern of
domain organization of NR-PKSs is SAT, KS, AT, PT, ACP, and
belonging to the b-lactamase superfamily, thus TE domains, from the N- to C-terminal end (Cox, 2007). The TE
domain, usually located at the C-terminal end of NR-PKSs, cata-
lyzes the last step to release products from the enzymes by
appears to be a novel type of thioesterase respon-
sible for product release in polyketide biosynthesis.
These findings show that ACAS synthesizes the scaf-
fold of atrochrysone carboxylic acid from malonyl-
CoA, and that ACTE hydrolyzes the thioester bond
between the ACP of ACAS and the intermediate to
release atrochrysone carboxylic acid as the reaction
product.
a Claisen cyclization of the polyketide intermediates, leading to
C-C bond formation, accompanied by cleavage of the thioester
bond between ACP and the polyketide intermediate (Fujii et al.,
2001). The TE domains of NR-PKSs contain a conserved Ser,
His, Asp catalytic triad, which is also common among the TE
domains of bacterial modular type I PKSs (Tsai et al., 2001)
and mammalian type I FASs (Chakravarty et al., 2004). The TE
domain of WAS in Aspergillus nidulans catalyzes a Claisen cycli-
zation of the second ring of a naphthopyrone, YWA (Fujii et al.,
2001). Site-directed mutagenesis of the conserved Ser and His
INTRODUCTION
Various fungal polyketides, including aflatoxin B1 (Minto and of the TE domain of WAS resulted in a switch from synthesis of
Townsend, 1997), melanin, tetrahydroxynaphthalene (Takano a naphthopyrone to synthesis of a citreoisocoumarin, a product
et al., 1995; Tsai et al., 1998), and lovastatin (Kennedy et al., derived from a lactone. Site-directed mutagenesis abolished the
1999), illustrate the diversity of chemical structures and the Claisen cyclization catalysis by this TE domain and resulted in
diverse biosynthetic potential of fungi. These polyketides are lactonization,
synthesized by iterative type I PKSs, which have a single modular Such lactone production is a well-known PKS shunt reaction.
architecture with individual functional domains and catalyze
In some NR-PKSs, the TE domain is absent or substituted by
a series of polyketide-forming reactions iteratively in a similar other domains. The TE domain of PksCT in Monascus purpureus
way to mammalian fatty acid synthases (FASs).
is replaced by methyltransferase and thioester reductase
a nonenzymatic C-O bond-forming reaction.
The typical reaction catalyzed by a fungal PKS, consisting of domains (Shimizu et al., 2005). The thioester reductase domain
a b-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier of PksCT appears to be responsible for product release, due to
protein (ACP) domains, is as follows. The starter unit is trans- its similarity in amino acid sequence to the reductase domain of
ferred to the KS domain, and an extender unit, such as malonyl nonribosomal peptide synthetases (NRPSs), which catalyzes
thioester, is transferred to the ACP by AT domain. Then, the product release from the NRPSs. Several known NR-PKSs do
KS domain catalyzes a decarboxylative Claisen condensation not contain TE or apparently any other domain responsible for
between an acyl thioester and an extender unit, resulting in the product release. How such TE-less NR-PKSs release their
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Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
Figure 1. Domain Organization of ACAS
(ATEG_08451) and ORFs near ATEG_08451
(A) Domain organizations of PksA in Aspergillus
parasiticus (AAS66004) and ACAS (ATEG_08451)
in A. terreus.
(B) ORFs in the vicinity of ATEG_08451.
(C) Coomassie brilliant blue-stained SDS-PAGE
analysis of purified ACAS (lane 1) and ACTE
(lane 2), together with molecular size markers.
In this report, we show that the TE-less
NR-PKS, ATEG_08451, produces several
anthraquinones in collaboration with a
physically discrete thioesterase, ATEG_
08450, by in vitro reconstitution of the
two enzymes and their heterologous
expression in A. oryzae. ATEG_08450,
sharing no apparent homology with the
classical TE domain of type I PKSs,
belongs to the b-lactamase superfamily
(Daiyasu et al., 2001). ATEG_08451 cata-
lyzes the formation of the scaffold of atroc-
hrysone carboxylic acid, and ATEG_08450
catalyzes hydrolysis of the thioester
between the ACP domain and the polyke-
tide intermediate. Thus, we named
ATEG_08451 ‘‘atrochrysone carboxylic
acid synthase’’ (ACAS) and ATEG_08450
‘‘atrochrysonecarboxylACPthioesterase’’
product is not well understood. Recently, Szewczyk et al. (2008) (ACTE). This is the first in vitro demonstration that a b-lactamase-
showed, by gene disruption experiments in the asperthecin like thioesterase releases the product from a TE-less type I PKS.
biosynthesis gene cluster in A. nidulans, that a TE-less NR-PKS
member of this gene cluster required a b-lactamase-like protein RESULTS
to synthesize asperthecin. They suggested that the b-lacta-
mase-like protein was responsible for releasing the product Domain Organization of ACAS
from the TE-less type I PKS. However, the role of this b-lacta- On the basis of the partial nucleotide sequences for the KS and AT
mase-like protein in product release from a TE-less NR-PKS domains of the emodin anthrone PKS in A. terreus RED1 (Couch
needs further investigation.
and Gaucher, 2004), we searched the genomic sequence of
Anthraquinones are a large family of compounds isolated from A. terreus NIH2624 for candidate emodin anthrone PKS genes
plants, fungi, lichens, and insects. Laxative effects are a well- in this strain using a BLAST search (Altschul et al., 1990).
known bioactivity of some natural anthraquinones. Recently, ATEG_08451, sharing 97% identity in amino acid sequence with
anthraquinones have been reported to possess some anticancer theemodinanthronePKS instrainRED1, wasfound. Asdescribed
properties, such as inhibiting cellular proliferation, inducing below, ATEG_08451 was confirmed to be responsible for the
apoptosis, and preventing metastasis (Huang et al., 2007). formation of atrochrysone carboxylic acid and was named ACAS.
Emodin is a representative anthraquinone, present in the roots
The domain search program (National Institute of Immunology)
and barks of many plants (Huang et al., 2007) and in several predicted that ACAS consisted of SAT, KS, AT, PT, and ACP
fungi, including Dermocybe, Cladosporium, Penicillium, and domains, from the N to C terminus (Figure 1A). Interestingly,
Aspergillus (Wells et al., 1975). The PKS (emodin anthrone ACAS lacked a TE domain.
PKS) that is involved in the biosynthesis of emodin anthrone in
A PKSwhoseTEdomainisinactivatedorremovedcannotcata-
A. terreus RED1 was identified during gene disruption experi- lyze a Claisen cyclization and thus yields a product with a lactone
ments to eliminate the production of sulochrin, an undesirable ring (Fujii et al., 2001); nevertheless, a plausible emodin biosyn-
cometabolite in the lovastatin fermentation by this fungus thetic pathway should include aldol cyclization of the C ring, not
(Couch and Gaucher, 2004).
lactonization, in addition to formation of the A and B rings by an
We studied ATEG_08451, a homolog of the emodin anthrone aldol cyclization and hydrolysis of the thioester bond between
PKS in A. terreus NIH2624. Interestingly, a domain search the ACP and the product (Figure 2B). Comparison of this pathway
showed that ATEG_08451 was a NR-PKS containing no TE with that for norsolorinic acid anthrone catalyzed by PksA (Minto
domain. Thus, it was a mystery as to how the TE-less enzyme, and Townsend, 1997; Figure 2A), a well-studied NR-PKS having
ATEG_08451, released its products.
a domain organization of NR-PKS, SAT, KS, AT, PT, ACP, and
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Product Release from PKS by a b-Lactamase-Type TE
Figure 2. The Route Proposed for Biosynthesis of Anthraquinones by the Actions of ACAS and ACTE
(A) Ring closure by PksA. The PT domain of PksA catalyzed the closure of the A and B rings, and the TE domain catalyzed the closure of the C ring by a Claisen
cyclization, resulting in the formation of norsolorinic acid anthrone.
(B) In vitro reactions proposed for ACAS and ACTE. ACAS catalyzes the closure of the A and B rings of atrochrysone carboxylic acid, and the C ring was presum-
ably cyclized non-enzymatically. ACTE catalyzed the hydrolysis of the thioester bond in the atrochrysone carboxyl ACP, resulting in product release. Conse-
quently, ACAS and ACTE produce compounds 1 to 6, in vitro. Endocrocin anthrone (2) and emodin anthrone (4) auto-oxidize in vitro to form endocrocin (1)
and emodin (5), respectively. Although 6 (m/z 257 [M-H]ꢀ) was predicted to be derived from a heptaketide intermediate judging from their m/z values, the structure
of 6 remained unidentified (C). Dimerization of endocrocin anthrone (2) in A. oryzae. Compound 7 is probably a mixture of enantiomers (10R, 100R) and (10S, 100S)-
endocrocin anthrone homodimer. Compound 8 is a meso form (10R, 100S)-endocrocin anthrone homodimer. Dimerization of 2 is probably catalyzed by an endog-
enous enzyme in A. oryzae.
TE, suggested that the PT domain of ACAS was involved in the acidanthrone is catalyzedby theTE domain viaa Claisenconden-
closure of the A and B rings of emodin because the PT domain sation. However, howC ringformationinemodin occursisunclear
of PksA catalyzes the formation of the A and B rings of this due to the absence of a TE domain. Additionally, how thioester
anthrone (Crawford et al., 2008). The C ring closure of norsolorinic bond cleavage occurs in emodin synthesis remains unknown.
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Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
Figure 3. HPLC Analysis of products from
in vitro reactions catalyzed by ACAS and
ACTE
All reactions contained malonyl-CoA. Boiled ACAS
(A) and ACAS (B) produced no product. Coincuba-
tion of ACAS and ACTE in the presence of malonyl-
CoA yielded products 1 to 6 (C).
In Vitro Reactions of ACAS
We first checked whether ACAS synthe-
sized the emodin scaffold in vitro. Our
attempts to produce an active ACAS
enzyme using various expression systems
in Escherichia coli failed for unknown
reasons. We then used an A. oryzae host-
vector system, in which the ACAS cDNA
was inserted between the amyB promoter
and terminator in plasmid pTAex3, which
was then integrated (several copies) into
the chromosome. The plasmid pTAex3-
ACAS-Nhis could direct the synthesis of
the ACAS enzyme as Met-Glu-His6-
ACAS. The ACAS protein with a His-tag
was thus produced in A. oryzae and puri-
fied by nickel-nitrilotriacetic acid (Ni-NTA)
affinity chromatography. Purified ACAS
gave a single major protein band of 195
kDa on SDS-PAGE (Figure 1C). Incubation
of the purified ACAS in the presence of
malonyl-CoA alone gave no product
(Figures 3A and 3B).
The presence of [2-14C]malonyl-CoA in
the reaction mixture led to 14C-labeling of
ACAS, showing that the AT domain of the
ACAS protein was active and that the
ACP domain was in the holo form (data
not shown). We assumed that the polyke-
tide intermediate remained attached to
A Claisen cyclization, catalyzed by a TE domain, includes the the ACP domain due to the absence of any TE domain in
cleavage of the thioester bond between ACP and the polyketide ACAS. Alkaline hydrolysis of the probable ACAS-polyketide
intermediate in addition to C ring formation, which occurs via the complex released product 1 and a trace amount of a putative
attack of the carbanion on the carbon atom adjacent to ACP. nonaketide (m/z 355 [M-H]ꢀ), as determined by liquid-chroma-
However, the aldol cyclization that is required for the C ring forma- tography atmospheric pressure chemical ionization mass spec-
tion in emodin is not accompanied by the cleavage of the thioester trometry (LC-APCIMS) analysis (see Figure S1 available online).
bond. Thus, to complete emodin formation by ACAS after C ring Product 1 was identified as endocrocin by comparing its reten-
closure, hydrolysis of the thioester bond is still required. These tion time (RT), ultraviolet irradiation (UV), mass spectrometry
observations suggested that ACAS cannot release its products (MS), and MS/MS spectra with those of the chemically synthe-
alone and that an additional enzyme might be responsible for sized authentic samples (Figure S2).
the hydrolysis of the thioester bond in these fungi.
Because nonenzymatic cyclization or dehydration of the inter-
Apart from the TE domain, the SAT domain of ACAS also mediate can occur during hydrolysis at an alkaline pH, it was
deserves mention. The SAT domain transfers acyl moieties, as unclear whether endocrocin itself was attached to ACAS. Our
starter substrates, to ACP. The SAT domain of ACAS does not attempts to release and detect the product produced by ACAS
contain the conserved GXCXG (X = any amino acid) active motif by incubating the ACAS-polyketide complex in the presence of
(Crawford et al., 2006); instead, it is replaced by a GXGXG hydroxylamine at physiological pH (pH 7.5) or in ethanol failed,
sequence. The SAT domain might thus be inactive, and the AT giving no product, presumably because the amount was below
domain could be responsible for both starter and extender unit the detection limit.
loading. In this study, however, we did not characterize the
SAT domain further.
C ring formation by ACAS was thus not confirmed by these
experiments. However, the octaketide, from which endocrocin
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Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
was derived, and the nonaketide detected were likely produced pTAex3 and ACTE in pPTRTA; in both plasmids, the cDNAs were
by ACAS. These findings suggested that recombinant ACAS under the control of the amyB promoter, and they integrated
itself could catalyze loading and condensation of several ma- (several copies) in the chromosome in the same cell. HPLC
lonyl-CoAs but could not release its product. We thus assumed and LC-APCIMS analyses of an ethyl acetate extract of the
that ACAS required an additional factor or enzyme to cleave the culture broth prepared from the A. oryzae transformant carrying
thioester bond between ACP and its intermediates.
both cDNAs revealed the production of several products, 1–10,
whereas the transformants carrying either cDNAs gave almost
no new product (Figures 4A–4D). Products 1 to 6 were also de-
In Vitro Reactions of ACAS and ACTE
We focused on ATEG_08450, which is located upstream of tected in the above-described in vitro reaction (Figure 3C); they
ACAS, in the opposite orientation on the chromosome were identified as endocrocin (1); compounds (2 and 3), derived
(Figure 1B), as a candidate for the thioesterase able to release from an octaketide; emodin anthrone (4); emodin (5); and
the product by cleaving the thioester bond between the ACP of compound (6), derived from a heptaketide. Products 7–10
ACAS and the polyketide intermediate. ATEG_08450 contains were newly detected in A. oryzae.
a metal-binding domain (THXHXDH; X = any amino acid) that is
Product 3, which was prepared in large amounts, was identi-
conserved in the family of metallo-b-lactamases (Crowder fied as atrochrysone by comparing its 1H nuclear magnetic reso-
et al., 1996; Concha et al., 1996) and has 22% identity in amino nance (NMR) spectrum with reported data (Gill and Morgan,
acid sequence to human glyoxalase II, as determined by three- 2001). Products 7 (m/z 597 [M-H]ꢀ) and 8 (m/z 597 [M-H]ꢀ)
dimensional position-specific scoring matrix (3D-PSSM) anal- were identified as diastereomers of homodimer of endocrocin
ysis. Glyoxalase II is responsible for the detoxification of methyl- anthrone (2; Figure 2C) by 1H- and 13C-NMR, heteronuclear
glyoxal by hydrolyzing a thioester bond of S-D-lactoylglutathione multiple quantum correlation (HMQC), heteronuclear multiple
to form D-lactic acid and regenerate glutathione (Cameron et al., bond correlation (HMBC), and nuclear Overhauser effect corre-
1999). As described below, ATEG_08450 turned out to be the lated spectroscopy (NOESY) analyses. The details of the NMR
thioesterase, as expected, and was named ACTE.
data are described in Figure S4. HMBC analysis of 7 and 8 re-
We produced ACTE as a His-tagged protein with the structure vealed a 1H-13C coupling between H10 and C100, indicating
Met-Asn-His-Lys-Val-His6-ACTE in E. coli, and purified it by the presence of a C10-C100 bond joining two monomers of endo-
affinity chromatography with the TALON metal affinity resin. crocin anthrone. Nuclear Overhauser effect (NOE) couplings
The SDS-PAGE pattern of the purified ACTE is shown in between H11 and H70 and between H4 and H50 were detected
Figure 1C. The addition of ACTE to the reaction mixture, consist- for 8, although no such coupling was detected for 7. This differ-
ing of ACAS and malonyl-CoA, led to production of six com- ence in NOE couplings reflects the difference in the dimerization
pounds (1–6; Figure 3C), as determined by high-performance manner between 7 and 8. Presumably, 7 is a mixture of enantio-
liquid chromatography (HPLC) and LC-APCIMS analysis, mers, (10R, 100R) and (10S, 100S)-endocrocin anthrone homo-
whereas (as described above) no product was detected in the dimer. The NMR data of (10R, 100R) and (10S, 100S)-endocrocin
absence of ACTE. Products 1, 4, and 5 were identified as endo- anthrone homodimer were identical. These two enantiomers (7)
crocin (1), emodin anthrone (4), and emodin (5), by comparing could not be separated by our HPLC method. Product 8 was
their RT, UV, MS, and MS/MS spectra with authentic samples probably a meso form, (10R, 100S)-endocrocin anthrone homo-
(Figures S2 and S3). Products 2 (m/z 299 [M-H]ꢀ) and 3 (m/z dimer. We assume that endocrocin anthrone (2) was dimerized
273 [M-H]ꢀ) were predicted to be derived from an octaketide, by an endogenous enzyme of A. oryzae. Production of 7 and 8
and 6 (m/z 257 [M-H]ꢀ) was perhaps derived from a heptaketide, supported the idea that endocrocin (1) was synthesized through
judging from their m/z values. Thus, ACAS apparently required endocrocin anthrone (2). Products 9 (m/z 553 [M-H]ꢀ) and 10
ACTE to produce the emodin scaffold in vitro, perhaps with (m/z 553 [M-H]ꢀ) were likely diastereomers of the heterodimer
product release as a result of the thioesterase activity of ACTE. consisting of emodin anthrone (4) and endocrocin anthrone (2).
The chemical structure of the major product (3), atrochrysone, The homodimer of endocrocin anthrone (7+8) was produced in
is described below. Interestingly, the yields and the molar ratios the highest yield among the products in A. oryzae, followed by
of the products formed in vitro remained invariable in reactions endocrocin (1), atrochrysone (3), and emodin (5).
containing larger amounts of ACTE than ACAS. Furthermore,
the yields of the products decreased dramatically in a reaction in vitro was thus atrochrysone (3) (molar ratio 1:3:5 = 1:8:0.1),
containing a lower concentration of ACTE than ACAS.
whereas the main product in vivo in A. oryzae was a homodimer
The main product resulting from the action of ACAS and ACTE
The presence of [1-14C]acetyl-CoA as a starter substrate and of endocrocin anthrone (7 + 8; molar ratio 1:3:5: 7 + 8 = 1:1:0.6:8;
nonlabeled malonyl-CoA as an extender substrate in the reaction Table 1). These findings suggest that ACAS and ACTE produce
mixture containing both ACAS and ACTE gave no radiolabeled endocrocin anthrone as the major product in A. oryzae (dis-
product, indicating that ACAS did not accept acetyl-CoA as cussed below). Detection of atrochrysone (3) and endocrocin
a starter. Such an inability to use acetyl-CoA as a starter was (1) suggested that atrochrysone carboxylic acid was a direct
also observed for PKS1 in Colletotrichum lagenarium (Fujii product of ACAS and ACTE (Figure 2B). Atrochrysone carboxylic
et al., 2000) and in PKS4 in Gibberella fujikuroi (Ma et al., 2007). acid was unstable and converted readily to atrochrysone (3) or
endocrocin anthrone (2), as described in the Discussion.
Analysis of ACAS and ACTE Reactions
The introduction of ACAS alone into A. oryzae gave endocro-
cin (1) and emodin (5) in trace amounts (data not shown).
by Heterologous Expression in A. oryzae
To prepare product 3 in large amounts for structural elucidation, Because the coexistence of ACTE and ACAS in A. oryzae
both ACAS and ACTE were expressed in A. oryzae. ACAS was in induced a 100-fold increase in the yields of the products, we
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Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
Figure 4. HPLC Analysis of Ethyl Acetate
Extracts Prepared from A. oryzae Trans-
formants
The transformants harboring pPTR I and pTAex3
(A), pPTRTA-ACTE, and pTAex3 (B) produced
no detectable compound. The transformants
harboring pPTR I and pTAex3-ACAS (C) produced
trace amounts of 1 and 5 (data not shown). The
transformant harboring pPTRTA-ACTE and
pTAex3-ACAS (D) produced compounds 1 to 10.
Compounds 9 and 10 were not separated in the
HPLC analysis, but were clearly separated in
LC-APCIMS analysis (data not shown).
ACTE as a Thioesterase
Responsible for Product Release
from ACAS
All the above findings suggested the
involvement of ACTE in the release of
atrochrysone carboxylic acid as the
product from ACAS after cleavage of the
thioester bond between the ACP of
ACAS and the octaketide intermediate
(Figure 2B). The expected thioesterase
activity of ACTE was tested using a
synthetic analog of the ACP-tricyclic
octaketide intermediate anthraquinone-
2-carboxylic
acid-N-acetylcysteamine
(Ac-NAC; 11) as a substrate (Figure 5A).
NAC is a mimic of the terminal portion of
phosphopantetheine, and NAC thioesters
have been widely used as ACP thioester
analogs. HPLC analysis of the reaction
mixture consisting of ACTE and Ac-NAC
revealed the production of anthraqui-
none-2-carboxylic acid (12; Figures 5B
and 5C), indicating that ACTE actually
catalyzed hydrolysis of the thioester
bond of Ac-NAC. Kinetic analysis showed
that ACTE gave 12 with a kcat of 24.5
0.9 sꢀ1 and a Km of 63.2 3.3 mM (Fig-
ure 5D). These data suggested that
ACTE catalyzed hydrolysis of the thio-
ester bond between the ACP of ACAS
and the octaketide intermediate, re-
leasing the corresponding acid as the
product.
We tested the ability of ACTE to hydro-
lyze an oxygen ester using 2-acetami-
doethyl-anthraquinone-2-carboxylate as
a substrate. This compound, having an
assumed that an endogenous ACTE homolog in this host cell oxygen ester, is structurally analogous to Ac-NAC. Incubation
served as a thioesterase to release atrochrysone carboxylic of ACTE with the compound under various conditions, however,
acid from ACAS. In fact, A. oryzae contains one ACTE homolog did not hydrolyze the oxygen ester. Thus, ACTE is unlikely to
(AO090701000530). Because the yields of the products of the generally cleave an oxygen ester.
in vitro reaction containing ACTE at a lower concentration than
We next examined the possible metal ion requirement of ACTE
ACAS were dramatically decreased (as described above), we for activity, because it belongs to the metallo-b-lactamase
assumed that ACTE was expressed at a higher level than ACAS. family. Metallo-b-lactamases are inactivated by chelating agents
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ACTE both in the in vitro reaction of malonyl-CoA and in the
in vivo expression in A. oryzae, we assumed that the direct
product was atrochrysone carboxylic acid (Figure 2B). Because
of the instability of atrochrysone carboxylic acid and its tendency
to readily convert to atrochrysone or endocrocin anthrone, atroc-
hrysone carboxylic acid was not detected throughout our study.
b-Keto acids are generally decarboxylated because they have
a low energy path for decarboxylation by way of the enol of the
product ketone (Guthrie, 2002). Because of this instability of
atrochrysone carboxylic acid, having a ketone group at the
b-position relative to the carboxyl group, decarboxylation occurs
readily, yielding atrochrysone. When dehydration of atrochry-
sone carboxylic acid occurs, instead of decarboxylation, the
observed product is endocrocin anthrone, which is unlikely to
be decarboxylated due to the absence of a ketone at the b-posi-
tion (Figure 2B). Emodin anthrone and emodin, both of which
were detected as products of ACAS and ACTE, were probably
derived from atrochrysone, not endocrocin, because the
carboxyl group of endocrocin is stable and acid treatment of
atrochrysone yielded emodin anthrone and emodin (data not
shown). This is consistent with the previous observation that
radiolabeled endocrocin was not incorporated into emodin in
Dermocybe, a fungus belonging to Basidiomycota (Steglich
et al., 1972). Homodimers of endocrocin anthrone were the
major products in A. oryzae carrying ACAS and ACTE, suggest-
ing that endocrocin anthrone is primarily produced in this heter-
ologous host. We suppose that a dehydratase, forming endocro-
cin anthrone, is present in A. oryzae. In A. terreus, it is unknown
whether atrochrysone carboxylic acid is converted to atrochry-
sone or to endocrocin anthrone. Although we attempted to
detect atrochrysone and endocrocin anthrone in the culture of
A. terreus NIH2624, no such compounds were detected.
Table 1. The Yields of the Compounds Produced by ACAS and
ACTE In Vitro and by Heterologous Expression in A. oryzae
In A. oryzae
In Vitro (nmol/120 mg)a (nmol/ml)b (mg/l)c
Endocrocin (1)
Atrochrysone (3)
Emodin (5)
1.00 0.06
7.66 0.13
0.135 0.01
17.3 3.5 5.43 1.10
15.8 1.3 4.33 0.36
9.83 1.51 2.65 0.41
58.1 9.4 34.7 5.6
Endocrocin anthrone ND
homodimer (7)
Endocrocin anthrone ND
67.5 11.0 40.4 6.6
homodimer (8)
Results are mean standard error (n = 3). ND, not detected.
a The yields of the compounds produced by ACAS and ACTE in vitro, per
120 mg ACAS.
b The yields of the compounds produced by ACAS and ACTE in A. oryzae,
per 1 ml broth.
c The yields of the compounds produced by ACAS and ACTE in A. oryzae,
per 1 l broth.
such as EDTA (Wang and Benkovic, 1998). Preparation of the
apoenzyme by vigorously dialyzing purified ACTE against a buffer
containing 10 mM EDTA resulted in precipitation of 65% of the
enzyme, as determined by measuring the protein concentration
in the soluble fraction. The initial velocity of the Ac-NAC hydro-
lysis by the soluble ACTE apoenzyme dropped by 96%. These
data clearly showed that ACTE required a metal ion for catalytic
activity and that ACTE was a metalloenzyme, like other b-lacta-
mases. We are currently studying the metal in ACTE.
DISCUSSION
Because endocrocin and atrochrysone were detected by LC- Because these compounds are metabolites at an early step in
APCIMS and HPLC analyses as major products of ACAS and the biosynthesis of geodin and asterric acid, atrochrysone might
Figure 5. Hydrolysis of a Thioester Bond
by ACTE
When anthraquinone-2-carboxylic acid-NAC (11)
was incubated with ACTE, anthraquinone-2-
carboxylic acid (12) was obtained (C) as a result
of the cleavage of the thioester bond as in the
scheme in (A). Boiled ACTE, as a negative control,
gave no product (B). Kinetic analysis of the
synthesis of 12 was performed in the presence of
11 at varying concentrations (D).
Chemistry & Biology 16, 613–623, June 26, 2009 ª2009 Elsevier Ltd All rights reserved 619

Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
be rapidly modified by tailoring modification enzymes (Couch has some structural similarity with human glyoxalase II, which
and Gaucher, 2004).
consists of a domain folding into a four-layered b sandwich
In the norsolorinic acid anthrone-producing reaction catalyzed and a domain that is predominantly a-helical. Glyoxalase II is
by PksA, thePTdomain catalyzedtheclosureof theAandB rings, a thioesterase in the glyoxalase system, which is responsible
followed by the Claisen cyclization of the C ring by the TE domain, for detoxification of methylglyoxal, a byproduct of metabolism,
resulting in product release (Crawford et al., 2008; Figure 2A). by hydrolyzing S-D-lactoylglutathione to form D-lactic acid and
Although the alkaline hydrolysis of ACAS after incubation in the regenerating glutathione (Cameron et al., 1999). It is a member
presence of malonyl-CoA gave only endocrocin and a putative of the metallo-b-lactamase superfamily and has a conserved
nonaketide product, the PT domain of ACAS is likely to catalyze metal-binding site, characteristic of this superfamily, as the
the closure of at least the A and B rings, like the PT domain of active site. Alignment of ACTE with members of glyoxalase II
PksA. If ACAS does not catalyze the closure of the A and B rings showed that ACTE has a metal-binding site (THXHXDH; X =
of the intermediate, various cyclization patterns of the interme- any amino acid) of the metallo-b-lactamase superfamily (Crow-
diate might yield many byproducts derived from the octaketide der et al., 1996; Concha et al., 1996; Figure S5). These bioinfor-
intermediate in addition to endocrocin. However, closure of the matics analyses suggested that ACTE belongs to the metallo-b-
C ring might occur nonenzymatically at an alkaline pH, because lactamase superfamily and has a structure different from a/b
aldol cyclization occurs readily under such conditions. At physio- hydrolase-fold and hotdog-fold thioesterases. Reflecting these
logical pH, however, it is unclear whether the C ring of endocrocin structural differences, ACTE is likely to catalyze the hydrolysis
is formed enzymatically or nonenzymatically. In the actinorhodin of the thioester between the ACP domain and the octaketide
biosynthesis in Streptomyces coelicolor A3(2), lactonization of intermediate, not cyclization, whereas the TE domains of itera-
a bicyclic intermediate is led by ActVI-ORF1. In the absence of tive type I PKSs catalyze the Claisen cyclization to release the
ActVI-ORF1, nonenzymatic aldol cyclization of the C ring occurs, products. This is consistent with the biosynthetic pathway
resulting in the formation of 3,8-dihydroxyl-1-methylanthraqui- proposed for asperthecin by Szewczyk et al. (2008). Further-
none-2-carboxylic acid, having the C ring of anthraquinone (Ichi- more, our study using a chelating agent, EDTA, suggested that
nose et al., 1999). Thus, it is likely that the C ring of atrochrysone ACTE requires a metal ion(s) for its activity. ACTE, belonging to
carboxylic acid is also closed by aldol cyclization nonenzymati- the b-lactamase superfamily, is apparently a metalloenzyme.
cally. We propose that the A and B rings of atrochrysone carbox- Taken together, ACTE is a novel type of thioesterase that is
ylic acid are cyclized by the PT domain of ACAS and that the C responsible for product release in the reaction of a type I PKS.
ring is cyclized nonenzymatically. The PT domain of PksA cata-
The presence of ACTE homologs is predicted in the clusters,
lyzes C2–C11 and C4–C9 cyclizations of the octaketide interme- including an open reading frame (ORF) for a TE-less NR-PKS in
diate (Figure 2A), whereas that of ACAS catalyzes C4–C13 and several filamentous fungi: five ORFs in Neosartorya fischeri,
C6–C11 cyclizations (Figure 2B). This regiospecificity of cycliza- three ORFs in Aspergillus fumigatus and A. nidulans, and one
tion might reflect their structural differences.
ORF in Pyrenophora tritici-repentis, Aspergillus niger, and A. or-
The TE domains of modular type I PKSs and type I FASs yzae (Figure S6). These ACTE homologs probably catalyze the
belong to the a/b hydrolase superfamily (Chakravarty et al., cleavage of the thioester bond between the TE-less type I
2004; Tsai et al., 2001). The core of the a/b hydrolase superfamily PKSs and resulting polyketides to release the products. In addi-
forms an a/b sheet consisting of eight b sheets connected by tion to iterative type I PKSs, some type II PKS clusters and
a helices (Ollis et al., 1992). The members contain a characteristic modular type I PKS clusters also have ACTE homologs. In the
Ser, His, Asp catalytic triad. The TE domains of iterative type I actinorhodin biosynthesis cluster, for example, ActIV shows
PKSs, with homology to those of FASs and containing this similarity with b-lactamases, including a similar metal-binding
´
conserved catalytic triad, appear to be members of the a/b domain (Fernandez-Moreno et al., 1992). The bacillaene biosyn-
hydrolase superfamily. Another type of TE includes SgcE10 thesis gene cluster in Bacillus subtilis also includes a b-lacta-
and NcsE10 that serve as physically discrete TEs for bacterial mase homolog, PksB (Butcher et al., 2007). We predict that
iterative type I PKSs SgcE, responsible for the biosynthesis of these homologs are involved in the polyketide biosynthesis as
C-1027 in Streptomyces globisporus, and NcsE, responsible a thioesterase for release of the product as ACTE in atrochrysone
for biosynthesis of neocarzinostatin in Streptomyces carzinosta- carboxylic acid biosynthesis.
ticus, both of which consist of KS, AT, ACP, KR, DH, and phos-
phopantetheinyl transferase domains and yield an enediyne core SIGNIFICANCE
(Zhang et al., 2008). These two thioesterases are similar to 4-hy-
droxybenzoyl-CoA thioesterase, which catalyzes the hydrolysis Fungal polyketides, most of which are synthesized by itera-
of the thioester of 4-hydroxybenzoyl-CoA in the degradation of tive type I PKSs, possess structural diversity and a variety of
4-chlorobenzoate. A homotetramer of the 4-hydroxybenzoyl- biological activities. However, only a few fungal PKS reac-
CoA thioesterase in Pseudomonas sp. strain CBS2 forms a tions have been characterized biochemically. The present
‘‘hotdog-fold’’ thioesterase structure, the monomer of which study showed that atrochrysone carboxylic acid synthase
consists of a long a helix wrapped by a five-stranded b sheet (ACAS) in A. terreus produces atrochrysone carboxylic
(Benning et al., 1998; Dillon and Bateman, 2004). ACTE is also acid as a direct product in collaboration with a physically
a discrete thioesterase that is responsible for the product release discrete thioesterase, atrochrysone carboxyl ACP thioester-
from ACAS, an NR-PKS. ACTE shows no significant homology ase (ACTE). ACTE has a conserved active site common to
with any TE domain of type I PKSs, type I FASs, or the hotdog- the b-lactamase superfamily and shows no similarity to the
fold thioesterase. Our 3D-PSSM analysis revealed that ACTE general TE domain of NR-PKSs. In vitro reconstitution of
620 Chemistry & Biology 16, 613–623, June 26, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
150 mM NaCl, and 10% glycerol. The ACTE protein gave a single protein
band at a position of ca. 37 kDa on SDS-PAGE (Figure 1C).
the reaction revealed the roles of ACAS and ACTE in the
synthesis of atrochrysone carboxylic acid. To our knowl-
edge, this is the first demonstration that a b-lactamase-
like thioesterase (ACTE) releases the product from a TE-
less NR-PKS (ACAS) in vitro. The present discovery can be
applied to ACTE homologs that are encoded in the vicinity
of the putative TE-less NR-PKS genes on chromosomes in
several filamentous fungi. These ACTE homologs presum-
ably release a product from the TE-less NR-PKSs by
cleaving the thioester bond between the ACP of the PKSs
and the polyketide intermediate. Furthermore, ACTE homo-
log genes are also found in type II and modular type I PKS
gene clusters in bacteria. Thus, we presume that an ACTE-
type thioesterase playing a role similar to ACTE in polyketide
biosynthesis is widely distributed in nature.
PKS Assay
The standard reaction mixture contained 0.13 mM malonyl-CoA, 50 mM
KH2PO4 (pH 7.5), 150 mMNaCl, 10%glycerol, 1 mMTris[2-carboxyethyl] phos-
phine, and 120 mg ACAS in a total volume of 760 ml. After incubation at 37ꢁC for
120 min, the reactions were quenched by the addition of 760 ml ice-cold 20%
(wt/vol) trichloroacetic acid. The pellet was resuspended in 500 ml 350 mM
KOH and incubated at 65ꢁC for 30 min to release covalently bound products.
After acidification with 50 ml 6 M HCl, the products were extracted twice with
500 ml ethyl acetate, and the organic layer was evaporated to dryness. The
residual material was dissolved in 20 ml methanol for LC-APCIMS analysis.
LC-APCIMS analysis in a negative mode was performed using the Agilent
1100 series (Agilent) with an ODS-80Ts reversed-phase HPLC column (2.0 3
150 mm; TOSOH) and the Esquire high-capacity trap plus system (Bluker Dal-
tonics), and the sample was elutedwitha linear gradient of 20%–60% CH3CN in
water (each containing 1% acetic acid) at a flow rate of 1 ml/min.
ACTE (21.8 mg) was additionally used in the standard reaction. The reaction
mixture was incubated at 37ꢁC for 120 min before being quenched with 76 ml
6 M HCl. The products were extracted with ethyl acetate and the organic layer
was evaporated to dryness. The residual material was dissolved in 20 ml meth-
anol for HPLC and LC-APCIMS analyses. LC-APCIMS analysis was carried out
as described above. HPLC analysis with an ODS-80Ts reverse-phase HPLC
column (2.0 3 150 mm; TOSOH) was carried out on a Hitachi LaChrom ELITE
system, and the sample was eluted with a linear gradient of 20%–60% CH3CN
in water (each containing 1% trifluoroacetic acid) at a flow rate of 1 ml/min. UV
spectra were detected on a Hitachi L-2450 diode array detector.
EXPERIMENTAL PROCEDURES
Materials
E. coli strains JM109, BL21(DE3) and plasmids pUC19, pT7blue, and pCold II,
pPTR I, restriction enzymes, T4 DNA ligase, Klenow enzyme, and PrimeSTAR
HS DNA Polymerase were purchased from Takara Biochemicals (Shiga,
Japan). The A. oryzae host-vector system pTAex3 and strain M-2-3 were
obtained from K. Gomi (Tohoku University). A. terreus NIH2624 was purchased
from the Fungal Genetic Stock Center (University of Kansas Medical Center,
Kansas City, KS). N-Acetylcysteamine was purchased from Aldrich, and ma-
lonyl-CoA and emodin were from Sigma. Anthraquinone-2-carboxylic acid
was purchased from TCI (Tokyo, Japan). [1-14C]Acetyl-CoA was purchased
from PerkinElmer Life Sciences (Boston, MA), and [2-14C]malonyl-CoA was
purchased from American Radiolabeled Chemicals (St. Louis, MO).
Syntheses of Emodin Anthrone and Endocrocin
Emodin anthrone was synthesized from emodin, as described by Falk et al.
(1993). The UV chromatogram, UV spectrum, MS/MS fragment pattern of
emodin anthrone are shown in Figure S3 with those of emodin as references.
Endocrocin was synthesized as described by Waser et al. (2005). The NMR
data, UV chromatogram, UV spectrum, and MS/MS fragment pattern of endo-
crocin are shown in Figure S2.
Construction of Plasmids
Please refer to Supplemental Experimental Procedures.
Production and Purification of Histidine-Tagged ACAS
Production and HPLC Analysis of Products of ACAS
The expression plasmid pTAex3-ACASNhis was linearized with PvuI and intro-
duced in the fungal host A. oryzae M-2-3 by the protoplast-polyethylene glycol
method (Gomi et al., 1987). Transformants were grown in Czapek-Dox medium
(0.3% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, 1% polypeptone)
with 2% glucose as a carbon source for 3 days and then transferred to Cza-
pek-Dox medium with 2% starch to induce the expression of ACAS under
the control of the amyB promoter. After 2 days of cultivation, mycelia were
ground in liquid nitrogen and suspended in buffer A (50 mM KH2PO4 [pH
7.5], 150 mM NaCl, 10% glycerol, 1 mM Tris[2-carboxyethyl]phosphine) and
Complete EDTA-free protease inhibitor cocktail (Roche). The mixture was
stirred at 4ꢁC for 30 min and subsequently centrifuged (13,000 g, 20 min).
The supernatant was filtered through Miracloth (Calbiochem). Ni-NTA super-
flow was added to the supernatant, and the solution was stirred at 4ꢁC for
1 hr. The protein/resin mixture was loaded into a gravity flow column, and
proteins were purified with a gradient of imidazole in buffer A. The purified histi-
dine-tagged protein was dialyzed against buffer A. The ACAS protein gave
a single protein band at a position of ca. 195 kDa on SDS-PAGE (Figure 1C).
and ACTE in A. Oryzae
The expression plasmid pTAex3-ACAS, linearized by PvuI, was used to trans-
form A. oryzae M-2-3 by the protoplast-polyethylene glycol method. pPTRTA-
ACTE, linearized by MunI, was then introduced in the transformant harboring
pTAex3-ACAS. Cotransformants were selected on Czapek-Dox pyrithiamine
(0.1 mg/ml) medium. Cotransformants were grown in Czapek-Dox medium
with 2% glucose as a carbon source for 3 days and then transferred to Cza-
pek-Dox medium containing 2% starch to induce the amyB promoter. Cultiva-
tion was continued for 3 days. The culture broth and cells were separated
through Miracloth. The broth was acidified to pH 3 with HCl and extracted
with ethyl acetate. The organic layer was evaporated to dryness. The residual
material was dissolved in 20 ml methanol for HPLC and LC-APCIMS analyses.
HPLC and LC-APCIMS analyses were carried out as described above.
Large Scale Preparation and Characterization of Products
of ACAS and ACTE
The culture conditions of A. oryzae carrying both ACAS and ACTE were the
same as those for the analytical scale, except that the culture was scaled up
to 2.4 l. After cultivation, broth and cells were separated through Miracloth.
The broth was acidified to pH 3 with HCl and extracted three times with ethyl
acetate. The organic layer was dried with Na2SO4 and evaporated to dryness.
The ethyl acetate extract was passed through a Sep-Pak Vac 20 cc (5 g) C18
cartridge (Waters) to remove lipid compounds. The crude material was dis-
solved in a small amount of chloroform/methanol (5:1, v/v) and flash-chroma-
tographed on silica gel using chloroform/methanol/acetic acid (98:2:1, v/v) as
the eluent. The eluates were evaporated and dissolved in methanol for reverse-
phase preparative HPLC, equipped with a DOCOSIL-B column (10 3 250 mm;
Senshu Scientific, Tokyo). The structure of 1 was determined by proton NMR
spectroscopy and HR-MS: 1H NMR (500 MHz, CDCl3) d 1.40 (s, 3H, C11H),
2.77 (dd, J = 17 Hz, 1H, C2H), 2.90 (d, J = 17 Hz, 1H, axial H of C2), 2.99
Production and Purification of Histidine-Tagged ACTE
E. coli BL21 (DE3) harboring pCold II-ACTE was precultured overnight in Luria-
Bertani (LB) medium containing 100 mg/ml ampicillin. The preculture was
transferred into LB medium containing 100 mg/ml ampicillin and incubated at
37ꢁC to an OD600 of 0.6. The cells were cultured further at 15ꢁC for 30 min
and then induced with 1 mM isopropyl thio-b-D-galactoside at 15ꢁC for
24 hr. The cells were harvested by centrifugation, resuspended in buffer con-
taining 50 mM KH2PO4 (pH 7.5), 150 mM NaCl, and 10% glycerol, and disrup-
ted by sonication. A crude cell-lysate was prepared by removal of cell debris
by centrifugation (10,000 g, 20 min). ACTE was purified using TALON Metal
Affinity Resin (Clontech), according to the manufacturer’s protocol. The puri-
fied histidine-tagged protein was dialyzed against 50 mM KH2PO4 (pH 7.5),
Chemistry & Biology 16, 613–623, June 26, 2009 ª2009 Elsevier Ltd All rights reserved 621

Chemistry & Biology
Product Release from PKS by a b-Lactamase-Type TE
(d, J = 16 Hz, 1H, equatorial H of C4), 3.08 (d, J = 16 Hz, 1H, axial H of C4), 3.30
(s, 1H, C3-OH), 6.38 (d, J = 2.5 Hz, 1H, C7H), 6.59 (d, J = 2.5 Hz, 1H, C5H), 6.83
(s, 1H, C10H), 9.84 (s, 1H, C8-OH). HR-MS (ESI): found for [C15H14O5-H]ꢀ,
273.07419. The structures of 7 and 8 were determined by proton and carbon
NMR spectroscopy with the aid of HMQC, HMBC, and NOESY analyses. The
NMR data of 7 and 8 are shown in Figure S4.
Received: December 1, 2008
Revised: April 2, 2009
Accepted: April 7, 2009
Published: June 25, 2009
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Fujii, I., Mori, Y., Watanabe, A., Kubo, Y., Tsuji, G., and Ebizuka, Y. (2000).
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Supplemental Data include seven figures and Supplemental Experimental
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chemistry-biology/supplemental/S1074-5521(09)00137-9.
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gillus oryzae with a plasmid containing the Aspergillus nidulans argB gene.
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T. Awakawa was supported by the Japan Society for the Promotion of
Science. We thank K. Gomi (Tohoku University) for providing us with the A. or-
yzae host-vector system. This work was supported by a grant from the New
Energy and Industrial Technology Development Organization of Japan and
a Grant-in-Aid for Scientific Research on Priority Area ‘‘Applied Genomics’’
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