Báo cáo khoa học: An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata Kiyofumi Wanibuchi1, Ping Zhang1,2, Tsuyoshi Abe1, Hiroyuki Morita3, Toshiyuki Kohno3, Guoshen Chen2, Hiroshi Noguchi1 and 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

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).

(EC 2.3.1.74)

triad [3–5]. The enzyme

enzyme-bound the of

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.

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The chalcone synthase (CHS) 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 reactions proceed through starter molecule loading, malonyl-CoA de- carboxylation, polyketide chain elongation, and cycli- intermediate. For zation example, CHS, a pivotal enzyme in flavonoid biosyn- thesis, catalyzes sequential condensation of the C6–C3

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serrata

unit 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 (Fig. 1A). The func- (4,2¢,4¢,6¢-tetrahydroxychalcone) 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 (Huperziaceae, (Thunb.) Trev. 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 the CHS to investigate the molecular evolution of 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.

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conserved sequences using primers based on the 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

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.

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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 Mr 45 831 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 identity with M. sativa CHS in the 30% (34 ⁄ 114) regions Met1–Asp24, His79–Ala99, Gln241–Asp261, Gly283–Ser304, Ser330–Ser343, and Gln358–Pro369

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(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). (Fig. 3) Phylogenetic tree analysis

Escherichia coli FABH (outgroup)

Mycobacterium tuberculosis PKS 18 Pseudomonas fuorescens PhlD (AAB48106)

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- [17], ALS from Rh. palmatum (Polygonaceae) 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- (5,7-dihydroxy-2-methyl- chromone), a pentaketide 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 Km ¼ 19.7 lm and kcat ¼ 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 (Km ¼ 36.1 lm and kcat ¼ 1.26 min)1) [20] and Rh. palmatum CHS1 (Km ¼ 61.1 lm and kcat ¼ 1.12 min)1) [13].

Bacteria

Streptomyces griseus RppA Streptmyces coelicolor A3(2) (CAC01488)

Mycobacterium tuberculosis PKS 10 Mycobacterium tuberculosis PKS 11 Deinococcus radiodurans (AAF11641)

Bacillus subtilis (AAA96613) Hypericum androsaemum BPS

Psilotum nudum VPS Huperzia serrata PKS1

Rheum palmatum ALS

Aloe arborescens OKS Aloe arborescens PCS

Plant

Ipomoea purpurea PKS-A (PCS) Ipomoea purpurea PKS-B (PCS)

Phalaenopsis sp. BBS

non-CHS

Ruta graveolens ACS Gerbera hybrida 2PS Hydrangea macrophylla CTAS Humulus lupulus VPS

Rheum palmatum BAS

Vitis vinifera STS

Arachis hypogaea STS

Medicago sativa CHS Pisum sativum CHS Phaseolus vulgaris CHS Pueraria lobata CHS

CHS / STS

Pinus strobus CHS Pinus sylvestris CHS

Pinus strobus STS

Pinus sylvestris STS

Ipomoea purpurea CHS-D Hydrangea macrophylla CHS Ipomoea purpurea CHS-E Petunia hybrida CHS Camellia sinensis CHS

Glycine max CHS Humulus lupulus CHS

Arabidopsis thaliana CHS Gerbera hybrida CHS

Rheum palmatum CHS1

Rheum palmatum CHS2

Ruta gravelolens CHS Oryza sativa CHS Zea mays CHS

Vitis vinifera CHS

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 synthase; BBS, bibenzyl synthase; BPS, benzophenone syn- thase; CHS, chalcone synthase; CTAS, 4-coumaroyltriacetic acid synthase; OKS, octaketides synthase; PCS, pentaketide chromone synthase; 2PS, 2-pyrone syn- thase; STS, stilbene synthase; VPS, valero- phenone synthase.

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Fig. 4. HPLC profile of Hu. serrata PKS1 enzyme reaction products from malonyl-CoA and (A) 4-coumaroyl-CoA, (B) 4-methoxycinnamoyl- CoA, (C) benzoyl-CoA, (D) phenylacetyl-CoA, (E) N-methylanthraniloyl-CoA, (F) hexanoyl-CoA, (G) acetyl-CoA, and (H) malonyl-CoA only. Note that with acid treatment, chalcones are nonenzymatically converted to racemic flavanones through a nonstereospecific ring-C closure.

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benzophenone (Fig. 4C) and phenylbenzylketone 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). (Fig. 4D), Interestingly,

Table 1. Enzyme reaction products from various starter substrates by Hu. serrata PKS1. Yield (%) under the standard assay condition (see Experimental procedures).

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Regular 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 (2,3¢,4,6-tetrahydroxybenzophe- into none) (2,4,6-tri- hydroxyphenylbenzylketone) respectively, along with triketide and tetraketide lactones [21]. Fur- thermore, Hu. serrata PKS1 also accepted aliphatic isobutyryl, n-hexanoyl, n- CoA starters (isovaleryl, octanoyl, n-decanoyl, and n-dodecanoyl) and yielded phloroglucinols (tetraketides) as well as triketide and produce 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 4-methoxy-2¢,4¢,6¢-tri- malonyl-CoA to

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in the case of

(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 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.

a

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- the CHS superfamily type III PKSs miscuity of requires a cautious interpretation of the results. Fur- thermore, besides several Lycopodium alkaloids and the chemical constituents of Hu. serrata terpenoids, 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- type III PKS that accepts bulky starter functional including 4-methoxycinnamoyl-CoA and substrates, 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

(Brea, CA, USA).

[2-14C]Malonyl-CoA (48 mCiÆmmol)1) and [1-14C]acetyl- CoA (47 mCiÆmmol)1) were purchased from Moravek Biochemicals 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].

Chemicals hydroxychalcone (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 tetraketide 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 cyclization optimally folded conformation in the pocket of the active site of the enzyme [20]. It is likely that Hu. serrata PKS1 has a larger starter substrate- binding pocket 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 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)-quinolone] [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 Km ¼ 15.9 lm and kcat ¼ 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 ⁄ Km value). sharing Interestingly, Hu. serrata PKS1,

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58% identity with Ru. graveolens amino acid sequence 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-methylanthraniloyl-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

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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.

cDNA 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 32 ¼ 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]: 112S ¼ 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¢, 368A ¼ 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.

30% MeOH;

5–17 min,

Then, 3¢-RACE, using two specific primers, 5¢-GAC AAA GTG CTG GCT GAA CCT CTG GAA TAC-3¢ and 5¢-GAA CCT CTG GAA TAC GTG CAT TTT CCC-3¢, 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.

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 30 (cid:2)C 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: 0–5 min, 30–60% MeOH; 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].

Expression of cDNA

A full-length cDNA was obtained using N- and C-terminal 5¢-CTG CTG GTC GAC ATG ACA AT- PCR primers: (the SalI C AAG GGA-3¢ site is underlined), and 5¢- CCG 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 D600 of 0.6 in LB medium containing 100 lgÆmL)1 of ampicillin at 23 (cid:2)C. Then, 1.0 mm isopropylthio-b-d-galactoside was added to induce protein expression, and the culture was incubated further at 23 (cid:2)C for 16 h.

327 nm. Triketide

Products of 4-methoxycinnamoyl-CoA

Flavanone (note that the chalcone produced was nonenzy- matically converted to flavanone) ) HPLC: Rt ¼ 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: (positive): MS, m ⁄ z 287 Rt ¼ 26.8 min. LC-ESI-MS [M + H]+, MS ⁄ MS (precursor ion at m ⁄ z 287), m ⁄ z 241 [M + H-CO2]+. UV: lac- kmax tone ) HPLC: Rt ¼ 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: kmax 359 nm.

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-

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Enzyme purification

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Products of N-methylanthraniloyl-CoA

References

1 Schro¨ der J (1999) The chalcone ⁄ stilbene synthase-type family of condensing enzymes. Comprehensive Natural Products Chemistry, Vol. 1, pp. 749–771. Elsevier, Oxford.

2 Austin MB & Noel JP (2003) The chalcone synthase

superfamily of type III polyketide synthases. Nat Prod Report 20, 79–110.

3 Ferrer JL, Jez JM, Bowman ME, Dixon RA & Noel JP (1999) Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat Struct Biol 6, 775–784.

Acridone ) HPLC: Rt ¼ 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: kmax 236 nm. Tetraketide lac- tone ) HPLC: Rt ¼ 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: kmax 235 nm. Triketide lactone ) HPLC: (positive): MS, m ⁄ z 218 Rt ¼ 26.9 min. LC-ESI-MS [M + H]+, MS ⁄ MS (precursor ion at m ⁄ z 218), m ⁄ z 176, 134. UV: kmax 263 nm. Quinolone ) HPLC: Rt ¼ 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.

4 Austin MB, Bowman ME, Ferrer J-L, Schro¨ der J &

Noel JP (2004) An aldol Switch discovered in stilbene synthases mediates cyclization specificity of Type III polyketide synthases. Chem Biol 11, 1179–1194.

5 Jez JM, Austin MB, Ferrer J, Bowman ME, Schro¨ der J & Noel JP (2000) Structural control of polyketide for- mation in plant-specific polyketide synthases. Chem Biol 7, 919–930.

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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-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 30 (cid:2)C for 20 min. The reaction products were extrac- ted and separated by TLC (Merck Art. 1.11798 Silica gel 60 F254, Merck, Darmstadt, Germany; ethyl acetate ⁄ hexane ⁄ AcOH ¼63 : 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 triplicates ± SD) using enzfitter software (average of (Biosoft, Cambridge, UK).

Enzyme kinetics

10 Lukacin R, Schreiner S, Silber K & Matern U (2001) Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae. Phytochemistry 66, 277–284.

11 Abe I, Takahashi Y, Morita H & Noguchi H (2001) Benzalacetone synthase. A novel polyketide synthase that plays a crucial role in the biosynthesis of phenyl- butanones in Rheum palmatum. Eur J Biochem 268, 3354–3359.

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].

Phylogenetic tree

Acknowledgements

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synthase superfamily of type III polyketide synthases from rhubarb (Rheum palmatum). Proc Japan Acad Series B 81, 434–440.

14 Abe I, Utsumi Y, Oguro S, Morita H, Sano Y &

Noguchi H (2005) A plant Type III polyketide synthase that produces pentaketide chromone. J Am Chem Soc 127, 1362–1363.

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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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Multifunctional type III PKS from Huperzia serrata

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of chalcone synthase, from Hydrangea macrophylla var. thunbergii. Eur J Biochem 263, 834–839.

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(2005) Engineered biosynthesis of plant polyketides: chain length control in an octaketide-producing plant Type III polyketide synthase. J Am Chem Soc 127, 12709–12716.

Substrate specificity of chalcone synthase: enzymatic formation of unnatural polyketides from synthetic cinnamoyl-CoA analogues. J Am Chem Soc 122, 11242–11243.

21 Morita H, Takahashi Y, Noguchi H & Abe I (2000)

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Enzymatic formation of unnatural aromatic polyketides by chalcone synthase. Biochem Biophys Res Commun 279, 190–195.

17 Liu B, Falkenstein-Paul H, Schmidt W & Beerhues L (2003) Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases. Plant J 34, 847–855.

22 Abe I, Watanabe T & Noguchi H (2004) Enzymatic for- mation of long-chain polyketide pyrones by plant type III polyketide synthases. Phytochemistry 65, 2447–2453. 23 Abe I, Abe T, Wanibuchi K & Noguchi H (2006) Enzy- matic formation of quinolone alkaloids by a plant type III polyketide synthase. Org Lett 8, 6063–6065.

18 Kreuzaler F & Hahlbrock K (1975) Enzymatic synthesis of aromatic compounds in higher plants. Formation of bis-noryangonin (4-hydroxy-6-[4-hydroxystyryl]2- pyrone) from p-coumaroyl-CoA and malonyl-CoA. Arch Biochem Biophys 169, 84–90.

24 Jez JM, Bowman ME & Noel JP (2002) Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity. Proc Natl Acad Sci USA 99, 5319–5324.

19 Akiyama T, Shibuya M, Liu HM & Ebizuka Y (1999) p-Coumaroyltriacetic acid synthase, a new homologue

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