polyketide synthase from Huperzia serrataKiyofumi Wanibuchi1, Ping Zhang1,2, Tsuyoshi Abe1, Hiroyuki Morita3, Toshiyuki Kohno3, Guoshen Chen2, Hiroshi Noguchi1and Ikuro Abe1,4 1 School o
Trang 1polyketide synthase from Huperzia serrata
Kiyofumi Wanibuchi1, Ping Zhang1,2, Tsuyoshi Abe1, Hiroyuki Morita3, Toshiyuki Kohno3,
Guoshen Chen2, Hiroshi Noguchi1and Ikuro Abe1,4
1 School of Pharmaceutical Sciences and the COE 21 Program, University of Shizuoka, Japan
2 Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, China
3 Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan
4 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
The chalcone synthase (CHS) (EC 2.3.1.74)
super-family of type III polyketide synthases (PKSs) are
pivotal enzymes in the biosynthesis of flavonoids as
well as a variety of plant secondary metabolites,
including stilbenes, benzophenones, acridones,
phloro-glucinols, resorcinols, pyrones, and chromones [1,2]
The type III PKSs of plant origin usually share 50–
75% amino acid sequence identity with each other,
and maintain a common three-dimensional overall fold with an absolutely conserved Cys-His-Asn cata-lytic triad [3–5] The enzyme reactions proceed through starter molecule loading, malonyl-CoA de-carboxylation, polyketide chain elongation, and cycli-zation of the enzyme-bound intermediate For example, CHS, a pivotal enzyme in flavonoid biosyn-thesis, catalyzes sequential condensation of the C6–C3
Keywords
acridone; chalcone synthase; Huperzia
serrata; type III polyketide synthase
Correspondence
I Abe, University of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
Fax ⁄ Tel: +81 54 264 5662
E-mail: abei@ys7.u-shizuoka-ken.ac.jp
(Received 30 October 2006, revised 15
December 2006, accepted 19 December
2006)
doi:10.1111/j.1742-4658.2007.05656.x
A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperzia-ceae) The deduced amino acid sequence of Hu serrata polyketide synthase
1 showed 44–66% identity to those of other chalcone synthase superfamily enzymes of plant origin Further, phylogenetic tree analysis revealed that
Hu serrata polyketide synthase 1 groups with other nonchalcone-produ-cing type III polyketide synthases Indeed, a recombinant enzyme expressed
in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloro-glucinols, and acridones In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malo-nyl-CoA to produce 4-methoxy-2¢,4¢,6¢-trihydroxychalcone and 1,3-dihyd-roxy-N-methylacridone, respectively In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site Although acridone alkaloids have not been isolated from Hu serrata, this is the first demon-stration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant Interestingly, Hu serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae)
Abbreviations
ACS, acridone synthase; ALS, aloesone synthase; BAS, benzalacetone synthase; BPS, benzophenone synthase; CHS, chalcone synthase; KPB, potassium phosphate buffer; OKS, octaketides synthase; PCS, pentaketide chromone synthase; PKS, polyketide synthase; STS, stilbene synthase; VPS, valerophenone synthase.
Trang 2unit of 4-coumaroyl-CoA with three C2 units from
malonyl-CoA, and this is followed by Claisen-type
cyclization of the enzyme-bound tetraketide
intermedi-ate, leading to the formation of naringenin chalcone
(4,2¢,4¢,6¢-tetrahydroxychalcone) (Fig 1A) The
func-tional diversity of the type III PKSs derives from a
small modification of the active site of the enzyme,
which greatly influences the selection of starter
sub-strate, the number of chain extensions, and the
mech-anisms of cyclization reactions
A primitive vascular plant, the Chinese club moss Huperzia serrata (Thunb.) Trev (Huperziaceae, recently reclassified by taxonomists, formerly Lycopo-dium serrata), is the famous medicinal plant that pro-duces the Lycopodium alkaloid huperzine A, a potent inhibitor of acetylcholinesterase and thus a promising drug for Alzheimer’s disease [6,7] In order to search for type III PKSs with novel catalytic functions, and
to investigate the molecular evolution of the CHS superfamily enzymes, we carried out PCR screening
A
B
C
D
Fig 1 Proposed mechanism for the conversion of (A) 4-coumaroyl-CoA and its analogs, (B) aromatic and aliphatic CoAs, (C) N-methylanthra-niloyl-CoA and (D) acetyl-CoA and malonyl-CoA by Hu serrata PKS1.
Trang 3using primers based on the conserved sequences
of known CHS enzymes Here we report a novel
type III PKS from the primitive vascular plant that
shows unusually versatile catalytic potential (Fig 1)
In particular, it is remarkable that the enzyme readily
accepted bulky starter substrates such as
N-methyl-anthraniloyl-CoA and efficiently produced
N-methylac-ridone, which is the first demonstration of enzymatic
formation of acridone by a type III PKS from a
non-Rutaceae plant Interestingly, Hu serrata PKS1
lacks most of the consensus active site sequences with
acridone synthase from Ruta graveolens (Rutaceae) [8–
10]
Results and Discussion
A cDNA encoding a novel type III PKS was cloned
and sequenced from young leaves of Hu serrata by
RT-PCR, using degenerate primers based on the
con-served sequences of known CHSs, as previously
des-cribed [11–15] The terminal sequences of cDNA were
obtained by the 3¢- and 5¢-RACE method A
full-length cDNA contained a 1197 bp ORF encoding an
Mr45 831 protein with 399 amino acids (the nucleotide sequence has been deposited in the EMBL⁄ DDBJ ⁄ GenBank databases under accession no DQ979827) The deduced amino acid sequence showed 44–66% identity
to those of other type III PKSs of plant origin: 66% (264⁄ 399) identity with Medicago sativa CHS [3], 52% (207⁄ 399) identity with Rheum palmatum aloesone syn-thase (ALS) [12], 53% (210⁄ 399) identity with Aloe ar-borescens pentaketide chromone synthase (PCS) [14], and 58% (231⁄ 399) identity with Ru graveolens acri-done synthase (ACS) [8] (Fig 2) Hu serrata PKS1 maintains most of the CHS active site residues, inclu-ding Met137, Gly211, Phe215, Gly216, Phe265, and Pro375, as well as the catalytic triad of Cys164, His303, and Asn336 (numbering in M sativa CHS) [3] Moreover, the active site Thr197, Gly256, and Ser338, the important residues for starter substrate selectivity and product chain length control [3,5,14,15], are also conserved in Hu serrata PKS1 However, despite these sequence similarities, Hu serrata PKS1 shares only 30% (34⁄ 114) identity with M sativa CHS in the regions Met1–Asp24, His79–Ala99, Gln241–Asp261, Gly283–Ser304, Ser330–Ser343, and Gln358–Pro369
Fig 2 Predicted secondary structure of Hu serrata PKS1 and sequence alignment with other CHS superfamily type III PKSs M.s CHS, Medicago sativa CHS; A.h STS, Arachis hypogaea stilbene synthase; R.p BAS, Rheum palmatum benzalacetone synthase; A.a OKS, Aloe arborescens octaketides synthase; R.g ACS, Ruta graveolens acridone synthase The catalytic triad (Cys164, His303, and Asn336), and the active site residues 197, 215, 256, 265 and 338 (numbering in M sativa CHS) are indicated by #, and residues for the CoA binding by + The consensus active site residues with Ru graveolens ACS [9,10] are indicated by ^ a-Helices (rectangles) and b-strands (arrows) of CHS are shown.
Trang 4(Fig 2) Homology modeling of Hu serrata PKS1
based on the sequence similarity with M sativa CHS
[3] did not reveal significant structural differences from
CHS (data not shown)
Phylogenetic tree analysis (Fig 3) revealed that
Hu serrata (Huperziaceae) PKS1 groups with other
nonchalcone-producing enzymes, including
valerophe-none synthase (VPS) from the primitive vascular plant
Psilotum nudum (Psilotaceae) [16], benzophenone
syn-thase (BPS) from Hypericum androsaemum
(Hyperia-ceae) [17], ALS from Rh palmatum (Polygona(Hyperia-ceae)
[12], and pentaketide chromone synthase (PCS) and
octaketides synthase (OKS) from Al arborescens
(Lili-aceae) [14,15] Notably, the latter three enzymes accept
acetyl-CoA (or malonyl-CoA) as a starter substrate to
produce a heptaketide
(2-acetonyl-7-hydroxy-5-methyl-chromone), a pentaketide
(5,7-dihydroxy-2-methyl-chromone), and octaketides (SEK4 and SEK4b),
respectively
Like other type III PKSs, recombinant Hu serrata PKS1 was functionally expressed in Escherichia coli with an additional hexahistidine tag at the N-terminus
As predicted from the high sequence similarity with regular CHS, the recombinant enzyme accepted 4-cou-maroyl-CoA as a good starter substrate, and efficiently yielded naringenin chalcone (4,2¢,4¢,6¢-tetrahydroxy-chalcone) after three condensations with malonyl-CoA (Figs 1A and 4A) In addition to chalcone, bisnoryan-gonin (a triketide) [18] and 4-coumaroyltriacetic acid lactone (a tetraketide) [19] were also formed as early released derailment byproducts For the chalcone formation reaction, Hu serrata PKS1 showed Km¼ 19.7 lm and kcat¼ 2.04 min)1 for 4-coumaroyl-CoA, with a pH optimum within a range of 7.0–8.0 The steady-state kinetics were better than those of the pre-viously reported Scutellaria baicalensis CHS (Km¼ 36.1 lm and kcat¼ 1.26 min)1) [20] and Rh palmatum CHS1 (Km¼ 61.1 lm and kcat¼ 1.12 min)1) [13]
Escherichia coli FABH (outgroup)
Mycobacterium tuberculosis PKS 18
Mycobacterium tuberculosis PKS 10 Mycobacterium tuberculosis PKS 11 Deinococcus radiodurans (AAF11641) Streptomyces griseus RppA
Bacillus subtilis (AAA96613) Streptmyces coelicolor A3(2) (CAC01488) Pseudomonas fuorescens PhlD (AAB48106)
Gerbera hybrida 2PS
Ruta graveolens ACS
Ipomoea purpurea PKS-A (PCS) Ipomoea purpurea PKS-B (PCS) Phalaenopsis sp BBS
Rheum palmatum BAS Vitis vinifera STS Humulus lupulus VPS
Rheum palmatum ALS Hypericum androsaemum BPS
Aloe arborescens OKS Aloe arborescens PCS
Huperzia serrata PKS1
Psilotum nudum VPS
Hydrangea macrophylla CTAS
Bacteria
Plant
non-CHS
CHS / STS
Petunia hybrida CHS
Gerbera hybrida CHS
Ruta gravelolens CHS Oryza sativa CHS Zea mays CHS Glycine max CHS
Arachis hypogaea STS
Pueraria lobata CHS Phaseolus vulgaris CHS
Medicago sativa CHS Pisum sativum CHS
Pinus strobus CHS Pinus sylvestris CHS Pinus strobus STS Pinus sylvestris STS Ipomoea purpurea CHS-D Ipomoea purpurea CHS-E Camellia sinensis CHS
Arabidopsis thaliana CHS Humulus lupulus CHS Hydrangea macrophylla CHS
Vitis vinifera CHS
Rheum palmatum CHS1 Rheum palmatum CHS2
0.1
Fig 3 Phylogenetic analysis of plant and bacterial type III PKS enzymes Multiple sequence alignment was performed with
CLUSTAL W (1.8) The indicated scale repre-sents 0.1 amino acid substitutions per site ACS, acridone synthase; ALS, aloesone syn-thase; BAS, benzalacetone synsyn-thase; BBS, bibenzyl synthase; BPS, benzophenone syn-thase; CHS, chalcone synsyn-thase; CTAS, 4-coumaroyltriacetic acid synthase; OKS, octaketides synthase; PCS, pentaketide chromone synthase; 2PS, 2-pyrone syn-thase; STS, stilbene synsyn-thase; VPS, valero-phenone synthase.
Trang 5Fig 4 HPLC profile of Hu serrata PKS1 enzyme reaction products from malonyl-CoA and (A) 4-coumaroyl-CoA, (B) 4-methoxycinnamoyl-CoA, (C) benzoyl-4-methoxycinnamoyl-CoA, (D) phenylacetyl-4-methoxycinnamoyl-CoA, (E) N-methylanthraniloyl-4-methoxycinnamoyl-CoA, (F) hexanoyl-4-methoxycinnamoyl-CoA, (G) acetyl-4-methoxycinnamoyl-CoA, and (H) malonyl-CoA only Note that with acid treatment, chalcones are nonenzymatically converted to racemic flavanones through a nonstereospecific ring-C closure.
Trang 6Regular CHS shows promiscuous substrate
specifici-ties in in vitro enzyme reactions [20–23] As in the case
of S baicalensis CHS, Hu serrata PKS1 also accepted
a variety of aromatic and aliphatic CoAs as starter
substrates, and produced aromatic tetraketides after
three condensations with malonyl-CoA (Table 1,
Fig 1) Thus, benzoyl-CoA and phenylacetyl-CoA
were also accepted as starter substrates, and converted
into benzophenone
(2,3¢,4,6-tetrahydroxybenzophe-none) (Fig 4C) and phenylbenzylketone
(2,4,6-tri-hydroxyphenylbenzylketone) (Fig 4D), respectively,
along with triketide and tetraketide lactones [21]
Fur-thermore, Hu serrata PKS1 also accepted aliphatic
CoA starters (isovaleryl, isobutyryl, hexanoyl,
n-octanoyl, n-decanoyl, and n-dodecanoyl) and yielded
phloroglucinols (tetraketides) as well as triketide and
tetraketide lactones (Fig 4F) [21,22] In most cases, the enzyme reactions afforded considerable amounts of the derailment lactone products, and a very small per-centage of aromatic minor products (Table 1) This is also the case for the previously reported type III PKSs, including S baicalensis CHS [20–22] and Rh palmatum CHSs [13] On the other hand, when incubated with acetyl-CoA (or malonyl-CoA only), Hu serrata PKS1 only produced triacetic acid lactone and tetra-acetic acid lactone [15] (Figs 1D and 4G,H)
Interestingly, further functional analyses revealed that Hu serrata PKS1 exhibits extraordinarily versatile catalytic potential For example, Hu serrata PKS1 accepted bulky 4-methoxycinnamoyl-CoA as a starter substrate, and carried out three condensations with malonyl-CoA to produce
4-methoxy-2¢,4¢,6¢-tri-Table 1 Enzyme reaction products from various starter substrates by Hu serrata PKS1 Yield (%) under the standard assay condition (see Experimental procedures).
Trang 7hydroxychalcone (Fig 4B) In contrast, as reported in
previous studies, regular CHS does accept
4-meth-oxycinnamoyl-CoA as a starter, but affords only a
tri-ketide and a tetratri-ketide lactone without formation of
a new aromatic ring system [20] For the regular CHS,
the steric and⁄ or electronic perturbations by the bulky
substituent at position 4 appeared to alter the stability
of the enzyme-bound tetraketide intermediate or the
optimally folded conformation in the cyclization
pocket of the active site of the enzyme [20] It is likely
that Hu serrata PKS1 has a larger starter
substrate-binding pocket (the so-called ‘coumaroyl-binding
pocket’ [3]) at the active site of the enzyme, and guides
the course of the Claisen-type aromatic ring formation
reaction of the enzyme-bound tetraketide intermediate
In particular, it is remarkable that Hu serrata PKS1
accepted N-methylanthraniloyl-CoA as a starter
substrate to produce N-methylacridone after three
condensations with malonyl-CoA (Figs 1C and 4E)
Interestingly, the enzyme also yielded diketide
quino-lone [4-hydroxy-1-methyl-2(1H)-quinoquino-lone] [23] as well
as triketide and tetraketide lactone byproducts [24] It
is known that anthranilic acid is a key intermediate in
the biosynthesis of acridone alkaloids, which occur in
greatest abundance in plants from the Rutaceae family
[8] In fact, ACS from Ru graveolens is the only
known type III PKS that selects
N-methylanthraniloyl-CoA as a starter to produce a tetraketide acridone
[8–10] Other type III PKSs, including regular CHS,
do not accept the bulky anthraniloyl starter, despite
their promiscuous substrate specificities, whereas the
M sativa CHS F215S mutant has been shown to
accept N-methylanthraniloyl-CoA to produce a
tet-raketide lactone, although without aromatic ring
for-mation reaction [24] Although acridone alkaloids have
not been isolated from Hu serrata [7], this is the first
demonstration of enzymatic production of acridone by
a type III PKS from a non-Rutaceae plant For the
acridone formation reaction, Hu serrata PKS1 showed
Km¼ 15.9 lm and kcat¼ 0.31 min)1 for
N-methyl-anthraniloyl-CoA, which represents a lower efficiency
than that of the naringenin chalcone formation (80%
decrease in the kcat⁄ Kmvalue)
Interestingly, Hu serrata PKS1, sharing 58%
amino acid sequence identity with Ru graveolens
ACS, lacks most of the active site residues, including
Ser132, Ala133, and Val265 (numbering in M sativa
CHS) (Fig 2), previously hypothesized to be
import-ant for the selection of N-methylimport-anthraniloyl-CoA as
a starter and for the formation of the acridone
scaf-fold [8–10] It has been reported that an ACS triple
mutant (S132T⁄ A133S ⁄ V265F) yielded an enzyme
that was functionally identical to CHS, whereas the
reverse mutations did not confer ACS specificity to CHS [9,10] Hu serrata PKS1 resembles CHS rather than ACS, and does not maintain the three residues (S132T⁄ A133S ⁄ V265F, as in the case of regular CHS), but shares ACS-like replacements at residues Lys85 and Ala98 (Fig 2) It is quite remarkable that, despite the apparent structural difference of the act-ive site, Hu serrata PKS1 exerts unusual and effi-cient acridone-producing activity In order to further elucidate the intimate structural details of the acri-done formation reaction, crystallization studies of
Hu serrata PKS1 are now in progress in our labor-atories
Finally, although the efficient production of naringe-nin chalcone by Hu serrata PKS1 suggests its in vivo function as a regular CHS, the physiologic relevance
of the in vitro utilization of the bulky substrates still remains to be determined Although relative enzyme activities observed in in vitro assays against a range of starters can help to narrow down the possible physio-logic function(s), the notorious in vitro substrate pro-miscuity of the CHS superfamily type III PKSs requires a cautious interpretation of the results Fur-thermore, besides several Lycopodium alkaloids and terpenoids, the chemical constituents of Hu serrata plant have not been well studied so far [6,7] To firmly establish the physiologic role of Hu serrata PKS1, fur-ther experiments are needed
In summary, Hu serrata PKS1 is a novel multi-functional type III PKS that accepts bulky starter substrates, including 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA Remarkably, this is the first demonstration of enzymatic formation of acridone scaffold by a type III PKS from a non-Rutaceae plant Further structural analyses of the enzyme promise to provide novel insights that will aid in understanding and engineering the substrate and product specificity
of the CHS superfamily type III PKSs
Experimental procedures
Chemicals
[2-14C]Malonyl-CoA (48 mCiÆmmol)1) and [1-14 C]acetyl-CoA (47 mCiÆmmol)1) were purchased from Moravek Biochemicals (Brea, CA, USA) 4-Coumaroyl-CoA, cinnamoyl-CoA, 4-methoxycinnamoyl-CoA, benzoyl-CoA, phenylacetyl-CoA and N-methylanthraniloyl-CoA were chemically synthesized as described previously [20–23] Malonyl-CoA, acetyl-CoA and other fatty acyl-CoA esters were purchased from Sigma (St Louis, MO, USA) Authen-tic samples of the enzyme reaction products were obtained
in our previous work [20–23]
Trang 8cDNA cloning
The Hu serrata plant used in this study was obtained from
L Hua (Zhejiang Academy of Medical Sciences, Hangzhou,
China) Total RNA was extracted from young leaves of
Hu serrata and reverse-transcribed using Reverscript
(Wako, Osaka, Japan) and oligo-dT primer (RACE 32¼
5¢-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT
TTT TTT T-3¢) The cDNA mixture obtained was used as
a template for the PCR reactions with inosine-containing
degenerate oligonucleotide primers based on the conserved
sequences of known CHSs, as described previously [11–15]:
112S¼ 5¢-(A ⁄ G)A(A ⁄ G) GCI ITI (A ⁄ C)A(A ⁄ G) GA(A ⁄ G)
TGG GGI CA-3¢, 174S ¼ 5¢-GCI AA(A ⁄ G) GA(T ⁄ C) ITI
GCI GA(A⁄ G) AA(T ⁄ C) AA-3¢, 368A¼ 5¢-CCC (C⁄ A)
(A⁄ T)I TCI A(A ⁄ G)I CCI TCI CCI GTI GT-3¢, and 380A ¼
5¢-TCI A(T ⁄ C)I GTI A(A ⁄ G)I CCI GGI CC(A ⁄ G) AA-3¢
(the number of each primer indicates the amino acid
num-ber of corresponding M sativa CHS) Nested PCR was
car-ried out with the 112S and 380A primer sets, and then with
174S and 368A to amplify a core 534 bp DNA fragment
Then, 3¢-RACE, using two specific primers, 5¢-GAC
AAA GTG CTG GCT GAA CCT CTG GAA TAC-3¢ and
was used to amplify a 365 bp DNA fragment; 5¢-RACE was
carried out using the Marathon cDNA Amplification kit
(Clontech, Mountain View, CA, USA) and two specific
prim-ers, 5¢-GAA AAT GCA CGT ATT CCA GA G GTT C-3¢
and 5¢-GTA TTC CAG AGG TTC AGC CAG CAT TTT
GTC-3¢, to amplify an 842 bp DNA fragment
Expression of cDNA
A full-length cDNA was obtained using N- and C-terminal
AT-C AAG GGA-3¢ (the SalI site is underlined), and
5¢-CCG 5¢-CCG CTG CAG TCA AAT GTT GAT ACT TCT-3¢
(the PstI site is underlined) The amplified DNA was
diges-ted with SalI⁄ PstI, and cloned into the SalI ⁄ PstI site of
pQE81L (Qiagen, Hilden, Germany) Thus, the
recombi-nant enzyme contains an additional hexahistidine tag at the
N-terminus After confirmation of the sequence, the
plas-mid was transformed into E coli M15 The cells harboring
the plasmid were cultured to a D600of 0.6 in LB medium
containing 100 lgÆmL)1 of ampicillin at 23C Then,
1.0 mm isopropylthio-b-d-galactoside was added to induce
protein expression, and the culture was incubated further at
23C for 16 h
Enzyme purification
The E coli cells were harvested by centrifugation at 3000 g
for 30 min using Kubota 5200 centrifuge (Kubota, Tokyo,
Japan) with RS-720 rotor, and resuspended in 40 mm
potas-sium phosphate buffer (KPB) (pH 7.9), containing 0.1 m NaCl Cells were disrupted by sonication, and centrifuged at
10 000 g for 30 min using a Kubota 3700 centrifuge with AF-5004 rotor The supernatant was passed through a col-umn of Ni Sepharose 6 Fast Flow (Amersham Bioscience, Piscataway, NJ, USA) After being washed with 20 mm KPB (pH 7.9), containing 0.5 m NaCl and 10 mm imidaz-ole, the recombinant enzyme was finally eluted with 15 mm KPB (pH 7.5), containing 10% glycerol and 500 mm imi-dazole Finally, to determine subunit composition, the puri-fied enzyme was applied to an HPLC gel filtration column (TSK-gel G3000SW, 7.5· 600 mm) (Tosoh, Tokyo, Japan), and was eluted with 100 mm KPB (pH 6.8), containing 10% glycerol and 0.2 m KCl at a flow rate of 1.0 mLÆmin)1
Enzyme reaction
The standard reaction mixture contained 54 lmol of star-ter-CoA, 108 lmol of malonyl-CoA and 20 lg of the puri-fied recombinant enzyme in a final volume of 500 lL of
100 mm KPB (pH 7.0) Incubations were carried out at
30C for 60 min, and stopped by adding 50 lL of 20% HCl The products were then extracted with 2000 lL of ethyl acetate, and analyzed by RP-HPLC on a TSK-gel ODS-80TsQA column (2.0· 150 mm) (Tosoh) with a flow rate of 0.8 mLÆmin)1, as described previously [11–15] For the standard assay, gradient elution was performed with
H2O and MeOH, both containing 0.1% trifluoroacetic acid:
17–25 min, 60% MeOH; 25–27 min, 60–70% MeOH; 27–
35 min, 70% MeOH; and 35–40 min, 70–100% MeOH Online LC-ESI-MS spectra were measured with an Agilent Technologies (Santa Clara, CA, USA) HPLC 1100 series coupled to a Bruker Daltonics (Bremen, Germany) esquire4000 ion trap mass spectrometer fitted with an ESI source as described previously [23] Identification of the enzyme reaction products was done by direct comparison with authentic compounds obtained in our previous work [15,20–23]
Products of 4-methoxycinnamoyl-CoA
Flavanone (note that the chalcone produced was nonenzy-matically converted to flavanone)) HPLC: Rt¼ 36.5 min LC-ESI-MS (positive): MS, m⁄ z 287 [M + H]+
, MS⁄ MS (precursor ion at m⁄ z 287), m ⁄ z 153 [M + H-C6H4OCH3
-C2H3]+ UV: kmax 287 nm Tetraketide lactone) HPLC:
Rt¼ 26.8 min LC-ESI-MS (positive): MS, m⁄ z 287 [M + H]+, MS⁄ MS (precursor ion at m ⁄ z 287), m ⁄ z 241 [M + H-CO2]+ UV: kmax 327 nm Triketide lac-tone) HPLC: Rt¼ 33.9 min LC-ESI-MS (positive): MS,
m⁄ z 245 [M + H]+
, MS⁄ MS (precursor ion at m ⁄ z 245),
m⁄ z 201 [M + H-CO2]+ UV: kmax359 nm
Trang 9Products of N-methylanthraniloyl-CoA
Acridone) HPLC: Rt¼ 32.1 min LC-ESI-MS (positive):
MS, m⁄ z 242 [M + H]+, MS⁄ MS (precursor ion at
m⁄ z 242), m ⁄ z 227, 200 UV: kmax236 nm Tetraketide
lac-tone) HPLC: Rt¼ 26.2 min LC-ESI-MS (positive): MS,
m⁄ z 260 [M + H]+
, MS⁄ MS (precursor ion at m ⁄ z 260),
m⁄ z 242, 134 UV: kmax235 nm Triketide lactone) HPLC:
Rt¼ 26.9 min LC-ESI-MS (positive): MS, m⁄ z 218
[M + H]+, MS⁄ MS (precursor ion at m ⁄ z 218), m ⁄ z 176,
134 UV: kmax263 nm Quinolone) HPLC: Rt¼ 24.2 min
LC-ESI-MS (positive): MS, m⁄ z 176 [M + H]+
, MS⁄ MS (precursor ion at m⁄ z 176), m ⁄ z 162, 134 UV: kmax 275,
315 nm
Enzyme kinetics
Steady-state kinetic parameters were determined by using
[2-14C]malonyl-CoA (1.8 mCiÆmmol)1) as a substrate The
experiments were carried out in triplicate using five
concen-trations (54.0, 43.2, 32.4, 21.6 and 10.8 lm) of
4-coumaroyl-CoA (or N-methylanthraniloyl-4-coumaroyl-CoA) in the assay mixture,
which contained 27 lm malonyl-CoA, 5 lg of purified
enzyme, and 1 mm EDTA, in a final volume of 500 lL of
100 mm Tris⁄ HCl buffer (pH 8.0) Incubations were carried
out at 30C for 20 min The reaction products were
extrac-ted and separaextrac-ted by TLC (Merck Art 1.11798 Silica gel 60
F254, Merck, Darmstadt, Germany; ethyl acetate⁄ hexane ⁄
AcOH¼63 : 27 : 5, v ⁄ v ⁄ v) Radioactivities were quantified
by autoradiography using a BAS-2000II bioimaging analyzer
(Fujifilm, Tokyo, Japan) Lineweaver–Burk plots of data
were employed to derive the apparent Km and kcat values
(average of triplicates ± SD) using enzfitter software
(Biosoft, Cambridge, UK)
Phylogenetic tree
In total, 50 amino acid sequences of CHS superfamily
enzymes were aligned, and the phylogenetic tree was
devel-oped with the clustal w (1.8) program (DNA Data Bank
of Japan, URL http://www.ddbj.nig.ac.jp) as described
pre-viously [11–13]
Acknowledgements
This work was supported by the PRESTO program
from the Japan Science and Technology Agency,
Grant-in-Aid for Scientific Research (nos 18510190
and 17310130), Cooperation of Innovative Technology
and Advanced Research in Evolutional Area (City
Area, the Central Shizuoka Area), and the COE21
program from the Ministry of Education, Culture,
Sports, Science and Technology, Japan
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