Cloning and structure-function analyses of quinolone- and acridone-producing novel type III polyketide synthases from citrus microcarpa

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 40, pp. 28845–28858, October 4, 2013

Cloning and Structure-Function Analyses of Quinolone- and

Acridone-producing Novel Type III Polyketide Synthases from

Citrus microcarpa^*

Received for publication, June 17, 2013, and in revised form, August 8, 2013 Published, JBC Papers in Press, August 20, 2013, DOI 10.1074/jbc.M113.493155

Takahiro Mori^‡, Yoshihiko Shimokawa^‡, Takashi Matsui^§, Keishi Kinjo^‡, Ryohei Kato^¶1, Hiroshi Noguchi^ʈ,

Shigetoshi Sugio^¶2, Hiroyuki Morita^§3, and Ikuro Abe^‡**⁴

From the ^‡Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the

^§Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, the ^¶Mitsubishi Chemical Group Science

and Technology Research Center Inc., 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502, the ^ʈSchool of Pharmaceutical

Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, and the **Japan Science and Technology Agency,

CREST, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

Background: Type III polyketide synthases (PKSs) synthesize various polyketide and alkaloid scaffolds.

Results: QNS synthesizes quinolone as the single product, whereas ACS produces acridone as the major product.

Conclusion: QNS and ACS are novel quinolone- and acridone-producing type III PKSs, respectively.

Significance: Structure-function analyses of QNS and ACS provide insights into molecular bases for alkaloid biosyntheses.

Two novel type III polyketide synthases, quinolone synthase of the bulky N-methylanthraniloyl-CoA within the catalytic

(QNS) and acridone synthase (ACS), were cloned from Citrus centers. However, the active site cavity volume of C. microcarpa

microcarpa (Rutaceae). The deduced amino acid sequence of C. ACS (760 Å³) is almost as large as that of M. sativa CHS (750 Å³),

microcarpa QNS is unique, and it shared only 56–60% identi- and ACS produces acridone by employing an active site cavity

ties with C. microcarpa ACS, Medicago sativa chalcone synthase and catalytic machinery similar to those of CHS. In contrast, the

(CHS), and the previously reported Aegle marmelos QNS. In cavity of C. microcarpa QNS (290 Å³) is significantly smaller,

contrast to the quinolone- and acridone-producing A. marmelos which makes this enzyme produce the diketide quinolone.

QNS, C. microcarpa QNS produces 4-hydroxy-N-methylquin- These results as well as mutagenesis analyses provided the

olone as the “single product” by the one-step condensation of first structural bases for the anthranilate-derived production of

N-methylanthraniloyl-CoA and malonyl-CoA. However, C. the quinolone and acridone alkaloid by type III polyketide

microcarpa ACS shows broad substrate specificities and pro- synthases.

duces not only acridone and quinolone but also chalcone,

benzophenone, and phloroglucinol from 4-coumaroyl-CoA,

The members of the chalcone synthase (CHS)⁵superfamily

of type III polyketide synthases (PKSs) are distributed in diverse

organisms, including plants, fungi, and bacteria, and are

responsible for the syntheses of various biologically and phar-

maceutically important natural products (1, 2). They are struc-

turally simple 40–45-kDa homodimeric enzymes that utilize a

conserved Cys-His-Asn catalytic triad in an active site to cata-

lyze the iterative condensations of C₂units derived from malo-

nyl-CoA to a CoA-linked starter molecule. The functional

diversity of the type III PKSs is basically derived from small

modifications within the active site architectures of the

enzymes, which influence the starter substrate selection, the

number of chain extensions, and the cyclization reaction mech-

anisms (1, 2). As one of the characteristic features of the type III

PKSs, the enzymes show broad substrate promiscuity and cat-

alytic versatility in in vitro reactions and convert structurally

and chemically distinct CoA starters into various molecular

scaffolds (2–6).

benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore,

the x-ray crystal structures of C. microcarpa QNS and ACS,

solved at 2.47- and 2.35-Å resolutions, respectively, revealed

wide active site entrances in both enzymes. The wide active site

entrances thus provide sufficient space to facilitate the binding

* This work was supported in part by grants-in-aid for scientific research from

the Ministry of Education, Culture, Sports, Science and Technology, Japan,

CREST, Japan Science and Technology Agency, and a grant from Suntory

for Bioorganic Research.

Theatomiccoordinatesandstructurefactors(code s 3WD 7 an d 3WD8)havebeendepos-

itedintheProteinDataBank(http://wwpdb.org/).

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/

GenBank^TM/EBI Data Bank with accession number(s) AB823730 and AB823699.

¹Present address: Medicinal Chemistry Research Laboratories II, Mitsubishi

Tanabe Pharma Corp., 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-

0033, Japan.

²Present address and to whom correspondence may be addressed: Separa-

tion Materials Laboratories R&D Center, Mitsubishi Chemical Corp., 1-1

Kurosaki-Shiroishi, Yahatanishi-ku, Kitakyushu, Fukuoka 806-0004, Japan.

Tel.: 81-93-643-2445; Fax: 81-93-643-2052; E-mail: sugio.shigetoshi@

mw.m-kagaku.co.jp.

The quinolone and acridone alkaloids have been investigated

as N-methyl-^D-aspartate (NMDA) and serotonin 5-hy-

³To whom correspondence may be addressed: Institute of Natural Medicine,

University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan. Tel.: 81-76-

434-7625; Fax: 81-76-434-5059; E-mail: hmortia@inm.u-toyama.ac.jp.

⁴To whom correspondence may be addressed: Graduate School of Pharma- ⁵The abbreviations used are: ACS, acridone synthase; BAS, benzalacetone

ceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-

0033, Japan. Tel.: 81-3-5841-4740; Fax: 81-3-5841-4744; E-mail: abei@

mol.f.u-tokyo.ac.jp.

synthase; CHS, chalcone synthase; PKS, polyketide synthase; QNS, quin-

olone synthase; r.m.s.d., root-mean-square deviation; RACE, rapid amplifi-

cation of cDNA ends.

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FIGURE 1. Proposed mechanism for the formation of alkaloids and polyketides by type III PKSs. Enzyme reaction products from malonyl-CoA and the

following: N-methylanthraniloyl-CoA (A), 4-coumaroyl-CoA (B), benzoyl-CoA (C), and hexanoyl-CoA (D). C. microcarpa QNS produces 4-hydroxy-N-methylquin-

olone as the sole product from N-methylanthraniloyl-CoA and triketide lactones from benzoyl-CoA and hexanoyl-CoA, whereas C. microcarpa ACS produces all

of the products.

droxytryptamine 3 receptor antagonists and as potential anti- ing CHS does not accept the bulky N-methylanthraniloyl-CoA

malarial drugs, respectively (7–9). The greatest abundance of starter (4, 11, 13–15), R. graveolens ACS can accept 4-couma-

these alkaloids is found in the Rutaceae plants, and N-methyl- royl-CoA as a starter substrate to produce naringenin chalcone

anthranilic acid is thought to be a key intermediate in their after three condensations with malonyl-CoA (Fig. 1B) (13, 15).

biosynthesis (10–12). Among the growing number of reported The other type III PKS is the recently reported quinolone syn-

type III PKSs, two kinds of type III PKSs specifically involved in thase (QNS) from Aegle marmelos, which produces the diketide

the biosynthesis of the anthranilate-derived alkaloids have been 4-hydroxy-N-methylquinolone by the one-step condensation

obtained from Rutaceae plants. One is acridone synthase (ACS) of N-methylanthraniloyl-CoA with one molecule of malonyl-

from Ruta graveolens, which catalyzes the iterative condensa- CoA (Fig. 1A) (12). The two type III PKS enzymes thus catalyze

tions of N-methylanthraniloyl-CoA with three molecules of the C–N bond-forming reactions, in addition to the C–C bond

malonyl-CoA to produce the tetraketide 1,3-dihydroxy-N- formation, to generate the anthranilate-derived alkaloid scaf-

methylacridone (Fig. 1A) (10, 11). Although the chalcone-form- folds. Notably, A. marmelos QNS does not produce the diketide

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Quinolone and Acridone Synthases from Citrus Microcarpa

quinolone specifically but also generates the tetraketide acri- CCC(C/A)(A/T)ITCIA(A/G)ICCITCICCIGTIGT-3Ј; and 380A ϭ

done from N-methylanthraniloyl-CoA in an in vitro enzyme 5Ј-TCIA(T/C)IGTIA(A/G)ICCIGG ICC(A/G)AA-3Ј (the

reaction (Fig. 1A). Thus, A. marmelos QNS could be regarded primer’s number indicates the amino acid number within M.

as another acridone-producing ACS, which simply yields the sativa CHS). Following the conditions described previously (4,

diketide quinolone as a by-product. This is analogous to the 19–21), nested PCR was performed with the primer sets 112S

tetraketide chalcone-forming CHS, which also produces and 380A and then with 174S and 368A to amplify the respec-

triketide and tetraketide lactone by-products in vitro (Fig. 1B). tive 550-bp core fragments of C. microcarpa QNS and ACS.

However, we previously demonstrated that benzalacetone syn- The 3Ј-RACE, using two gene-specific primers (209S, 5Ј-ACA-

thase (BAS) from Rheum palmatum, which normally produces TCTTGGTTGGGCAAGCTGTGTTTG-3Ј, and 252S, 5Ј-

the diketide benzalacetone by the one-step condensation of ACCAACAAGTTTATCAGGGCACACGTG-3Ј for QNS, and

4-coumaroyl-CoA with malonyl-CoA, also accepts N-methyl- 207S, 5Ј-GATTCTCTAGTGGGTCAGGCTCTTT-3Ј, and

anthraniloyl-CoA as a starter substrate to produce the quin- 245S, 5Ј-GCTTCTGCCTGATTCTGATGGTGCAA-3Ј for

olone by condensation with malonyl-CoA (Fig. 1B) (16).

ACS), and the oligo(dT) primer RACE32, was used to amplify the

We now report the first QNS that produces 4-hydroxy-N- QNS’s 472-bp cDNA and the ACS’s 642-bp fragment. The

methylquinolone from N-methylanthraniloyl-CoA, as well as 5Ј-RACE was performed using the 5Ј-RACE system (Invitrogen)

another acridone-producing ACS, from the leaves of the and two gene-specific primers (261A, 5Ј-TTTCACCTGTGTGA-

natural anti-inflammatory Rutaceae plant Citrus microcarpa. TAAACTTGTTGGT-3Ј, and 218A, 5Ј-CCAAACACAGCTTG-

Remarkably, in contrast to the previously reported quinolone- CCCAACCAAGATG-3Ј for QNS, and 253A, 5Ј-ATTGCACCA-

and acridone-forming A. marmelos QNS, C. microcarpa QNS TCAGAATCAGGCAGAAGC-3Ј, and 215A, 5Ј-AAAGAGCCT-

produces the diketide quinolone as the “single product” by the GACCCACTAGAGAATC-3Ј for ACS) to amplify QNS’s 643-bp

one-step condensation of N-methylanthraniloyl-CoA with and ACS’s 681-bp DNA fragments. The full-length cDNA encod-

malonyl-CoA (Fig. 1A). However, the newly obtained C. micro- ing M. microcarpa QNS was obtained using N- and C-terminal

carpa ACS shows quite promiscuous substrate specificities and primers as follows: 5Ј-GCGAGATCTATGGAATCAATGGCG-

produces not only the anthranilate-derived acridone and quin- AAAGTGAAAAATTTTCTTAATGCCAAG-3Ј(sense, the BglII

olone but also accepts various starter substrates to generate site is underlined) and 5Ј-CGCCTCGAGTCAACAGGCGGAA-

distinct molecular scaffolds, including chalcone, benzophe- TCAATAGGGACGCT-3Ј (antisense, the XhoI site is under-

none, and phloroglucinol (Fig. 1) (1, 2, 17). To clarify the mech- lined). The full-length cDNA encoding C. microcarpa ACS was

anistic details of the enzyme reactions, we also solved the x-ray finally obtained using N- and C-terminal-specific primers as fol-

crystal structures of C. microcarpa QNS and ACS, at 2.47- and lows: 5Ј-GCGAGATCTATGGTAACCATGGAGGAGATTAG-

2.35-Å resolutions, respectively. A comparison of the crystal AAGGGCT-3Ј (sense, the BglII site is underlined) and 5Ј-CGCC

structures revealed the unique active site architectures of these TCGAGTCATGCTTCTATAGGGAGACTGTGCAACA-3Ј

two enzymes and provided the first structural basis for the pro- (antisense, the XhoI site is underlined). The amplified full-length

duction of the anthranilate-derived quinolone and acridone C. microcarpa QNS and ACS cDNA fragments were digested with

alkaloids by type III PKSs.

BglII/XhoI and cloned into the BamHI/XhoI sites of pQE80L

(Novagen), respectively. Both recombinant enzymes thus contain

an additional N-terminal hexahistidine tag. The nucleotide

EXPERIMENTAL PROCEDURES

Materials—The [2-¹⁴C]malonyl-CoA (55 mCi/mmol) and sequence was determined by using an ABI Prism 3100 Genetic

[1-¹⁴C]acetyl-CoA (52.5 mCi/mmol) were purchased from Analyzer (Applied Biosystems).

Moravek Biochemicals (Brea, CA). The 4-coumaroyl-CoA and

Phylogenetic Tree—A total of 52 type III PKS amino acid

N-methylanthraniloyl-CoA were chemically synthesized as sequences were aligned, and a phylogenetic tree was developed

described previously (4, 18). Malonyl-CoA, hexanoyl-CoA, and with the ClustalW (1.8) program (DNA Data Bank of Japan), as

benzoyl-CoA were purchased from Sigma. Authentic samples reported previously (4).

of the enzyme reaction products were obtained in our previous

work (4, 18).

Expression and Purification of C. microcarpa QNS and

ACS—The plasmids containing the full-length cDNAs encod-

Gene Cloning of QNS and ACS—C. microcarpa Bunge leaves ing C. microcarpa QNS and ACS were individually transformed

were harvested from the Matakichi farm in Okinawa, Japan, into Escherichia coli M15. The cells harboring the plasmids

and immediately frozen in liquid nitrogen. Total RNA was were cultured to an A₆₀₀of 0.6 in LB medium containing 100 ␮g

extracted with an RNeasy plant mini kit (Qiagen) and was ml^Ϫ1ampicillin at 23 °C, and 1.0 mM isopropyl 1-thio-␤-D-ga-

reverse-transcribed using Superscript II RT (Invitrogen) lactopyranoside was then added to induce protein expression.

and the oligo(dT) primer (RACE 32 ϭ 5Ј-GGCCACGCGT- The culture was incubated further at 23 °C for 16 h. All of the

CGACTAGTACTTTTTTTTTTTTTTTTT-3Ј), according to following procedures were performed at 4 °C. The E. coli cells

the manufacturer’s protocol. The single strand cDNA thus were harvested by centrifugation at 5,000 ϫ g and resuspended

obtained was used as the template for the PCRs with inosine- in 50 m^MTris-HCl buffer (pH 7.5), containing 0.2 M NaCl, 5%

containing degenerate oligonucleotide primers, designed on (v/v) glycerol, and 5 m^Mimidazole (buffer A). The cells were

the basis of the conserved sequences of known CHSs, as disrupted by sonication, and the lysate was centrifuged at

described previously (4, 19–21): 112S ϭ 5Ј-(A/G)A(A/G)GCI- 12,000 ϫ g for 30 min. The supernatant was loaded onto a

ITI(A/C)A(A/G)GA(A/G)TGGGGICA-3Ј; 174S ϭ 5Ј-GCIAA- nickel-Sepharose 6 Fast Flow column (GE Healthcare) equili-

(A/G)GA(T/C)ITIGCIGA(A/G)AA(T/C)AA-3Ј; 368A ϭ 5Ј- brated with buffer A. After washing the resin with 50 m^MTris-

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Quinolone and Acridone Synthases from Citrus Microcarpa

HCl buffer (pH 7.5), containing 0.2 M NaCl, 5% (v/v) glycerol, PEG3350. Crystals of ACS were observed a few days later in a

and 10 m^Mimidazole (buffer B), the recombinant protein was

subsequently eluted with buffer B containing 300 m^Mimidaz-

ole. The protein solution was then diluted 5-fold with 50 m^M

Tris-HCl buffer (pH 7.5), containing 5% (v/v) glycerol and 2 mM

DTT (buffer C), and applied to a Resource-Q column (GE

Healthcare). The column was washed with buffer C containing

50 m^MNaCl, and the protein was subsequently eluted using a

linear gradient of 50–300 m^MNaCl. The protein solution was

concentrated to 5 ml, purified to homogeneity by gel filtration

chromatography on a Superdex 200 HR column (16/60 GL; GE

Healthcare), and concentrated to 10 mg/ml in 20 m^MTris-HCl

buffer (pH 7.5), containing 100 m^MNaCl and 2 mM DTT.

Enzymatic Reactions of C. microcarpa QNS and ACS—The

standard reaction mixture contained 54 ␮mol of starter CoA,

108 ␮mol of malonyl-CoA, and 20 ␮g of the purified recombi-

nant enzyme, in a final volume of 500 ␮l of 100 mM potassium

phosphate buffer (pH 7.0). The reactions were incubated at

30 °C for 60 min and stopped by adding 50 ␮l of 20% HCl. The

products were then extracted three times with 500 ␮l of

ethyl acetate, concentrated by evaporation, and analyzed by RP-

HPLC on a COSMOSIL௡ 5C18-MS-II column (4.6 ϫ 250 mm)

(Nacalai Tesque) at a flow rate of 0.8 ml/min, as described pre-

viously (4–6). Gradient elution was performed with H₂O and

MeOH, both containing 0.1% trifluoroacetic acid: 0–5 min,

30% MeOH; 5–17 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. On-line LC-ESI-MS spectra

were measured with an Agilent Technologies (Santa Clara, CA)

HPLC 1100 series coupled to a Bruker Daltonics (Bremen, Ger-

many) Esquire 4000 ion trap mass spectrometer fitted with an

ESI source, as described previously (4–6). The enzyme reaction

products were identified by direct comparisons to authentic

compounds.

crystallization solution consisting of 100 m^MHEPES-NaOH

(pH 7.5), 1,400 m^Mammonium sulfate, and 2 mM CoASH. Fur-

ther attempts to crystallize C. microcarpa QNS and ACS were

performed, using Additive Screen (Hampton Research), at var-

ious pH values, 18% PEG3350, 100 m^MHEPES-NaOH (pH 7.5),

and 1,400 m^Mammonium sulfate as a precipitant, respectively.

Diffraction quality crystals of C. microcarpa QNS were finally

obtained in 50 m^MTris-HCl (pH 7.0), 18% PEG3350, and 4%

(v/v) 1-propanol, and those of C. microcarpa ACS were finally

obtained in 100 m^MHEPES-NaOH (pH 7.5), 1,400 mM ammo-

nium sulfate, 2 m^MNiCl₂, and 2 mM CoASH using the sitting-

drop vapor diffusion method at 20 °C.

The crystals of C. microcarpa QNS were transferred into a

cryoprotectant solution, consisting of 50 m^MTris-HCl (pH 7.0),

18% PEG3350, 4% (v/v) 1-propanol, and 18% glycerol. After a

few seconds, the crystals were picked up in a nylon loop and

then flash-cooled at Ϫ173 °C in a nitrogen gas stream. X-ray

diffraction data sets were collected on beamline BL17A at the

Photon Factory (wavelength 0.9800 Å) using an ADSC Quan-

tum 315 CCD detector, with a distance of 250 mm between the

crystal and the detector. A total of 180 frames was recorded,

with a 1° oscillation angle and 1-s exposure time.

The crystals of C. microcarpa ACS were transferred into a

cryoprotectant solution, consisting of 100 m^MHEPES-NaOH

(pH 7.5), 1,400 m^Mammonium sulfate, 2 mM NiCl₂, and 20%

glycerol. After a few seconds, the crystals were picked up in a

nylon loop and then flash-cooled at Ϫ173 °C in a nitrogen gas

stream. X-ray diffraction data sets were collected on beamline

NW-12 of the Photon Factory-AR (wavelength 1.00000 Å)

using an ADSC Quantum 210 CCD detector, with a distance of

150 mm between the crystal and the detector. A total of 180

frames were recorded, with a 1° oscillation angle and 1-s expo-

sure time. The data were indexed, integrated, and scaled with

the HKL-2000 program package (22).

Enzyme Kinetics—Steady-state kinetic parameters were

determined using [2-¹⁴C]malonyl-CoA (1.8 mCi/mmol) as the

substrate. The experiments were performed in triplicate, using

five concentrations (51.2, 25.6, 12.8, 6.4, and 3.2 ␮M) of starter-

CoA and 4 ␮g of purified enzyme, in a final volume of 100 ␮l of

100 m^MTris-HCl (pH 8.0). The reactions were incubated at

30 °C for 30 min. The reaction products were extracted twice

with 100 ␮l of ethyl acetate and separated by TLC (Merck

1.11798 silica gel 60 F₂₅₄; ethyl acetate/hexane/AcOH ϭ

63:27:5, v/v/v). Radioactivities were quantified by autoradiog-

raphy, using a BAS-2000II bioimaging analyzer (Fujifilm,

Tokyo, Japan). Lineweaver-Burk plots of data were employed to

derive the apparent K_mand k_catvalues (average of triplicates Ϯ

S.D.) using the EnzFitter software (BIOSOFT, Cambridge, UK).

Crystallization and Structure Refinement of QNS and ACS—

Initial crystallization attempts and optimization of the crystal-

lization conditions for both enzymes were performed at 20 °C,

using the sitting-drop vapor diffusion method. First, 0.5 ␮l of

either the purified recombinant QNS or ACS protein and an

equal volume of reservoir solution were mixed and equilibrated

against 50 ␮l of reservoir solution, using a 96-condition crystal-

lization screen originally designed by Mitsubishi Chemical

Corp. Crystals of QNS appeared the next day in a crystallization

Structure Refinement—The initial phases of the C. micro-

carpa QNS and ACS structures were determined by molecular

replacement, using the M. sativa CHS structure (Protein Data

Bank code 1CGK) as the search model. Molecular replacement

was performed with MOLREP in the CCP4 suite (23, 24). The

structure was modified manually with Coot (25) and refined

with PHENIX (26). The final model of C. microcarpa QNS con-

sisted of residues 1–389 of monomers A–D, two molecules of

glycerol, and 198 molecules of water. The final model of C.

microcarpa ACS consisted of residues 1–389 with 11 artificial

residues, including an artificial Met as the initiation codon and

the N-terminal His₆tags of monomers A and B, two molecules

of CoA-SH, nine molecules of nickel, three molecules of SO₄,

and 331 molecules of water. The qualities of the final models

were assessed with MolProbity (27). A total of 97.1% of the

residues in the C. microcarpa QNS structure are in the favored

regions of the Ramachandran plot, and 2.9% are in the allowed

regions, although a total of 97.5% of the residues in the C. micro-

carpa ACS structure are in the most favored regions of the

Ramachandran plot, and 2.5% are in the allowed regions. The

coordinates and structure factors have been deposited in

solution consisting of 100 m^MTris-HCl (pH 7.0) and 18% the Protein Data Bank (Protein Data Bank code 3WD8 for the

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Quinolone and Acridone Synthases from Citrus Microcarpa

FIGURE 2. Sequence alignment of C. microcarpa QNS/ACS with other plant type III PKSs. CmQNS, C. microcarpa QNS; CmACS, C. microcarpa ACS; AmQNS, A.

marmelos QNS; RgACS, R. graveolens ACS; MsCHS, M. sativa chalcone synthase; HsPKS1, H. serrata PKS1; RpBAS, R. palmatum BAS. The catalytic triad (Cys-164,

His-303, and Asn-336) is colored red, and the active site residues, 132, 133, 137, 194, 197, 211, 215, 256, 265, 338, 375, are highlighted in blue (numbering in M.

sativa CHS).

RESULTS

C. microcarpa QNS apo structure and code 3WD7 for the C.

microcarpa ACS CoA-SH complexed structure).

Sequence Analyses of QNS and ACS—Two full-length cDNAs

encoding novel type III PKSs, QNS and ACS, were cloned and

sequenced from the leaves of C. microcarpa by the RT-PCR

method (the details of the method are described under “Exper-

imental Procedures”). The full-length QNS and ACS cDNAs

contained 1,191- and 1,176-bp open reading frames encoding

M_r43,331 and 42,830 proteins with 396 and 391 amino acids,

respectively (GenBank^TMaccession numbers AB823730 and

AB823699). Notably, the deduced amino acid sequence of C.

microcarpa QNS is quite unique and shared 60% identity to that

of the acridone-producing C. microcarpa ACS, 56% identity to

R. graveolens ACS (10), 59% identity to the quinolone-produc-

ing A. marmelos QNS (12), 60% identity to the chalcone-pro-

ducing Medicago sativa CHS (29), and 58% identity to the ben-

zalacetone-producing R. palmatum BAS (Fig. 2) (19). No

additional cDNAs encoding other type III PKS isomers were

obtained in this study.

The sequence analysis revealed that the C. microcarpa QNS

retains the CHS’s active site residues, such as Gly-211, Pro-375,

and the “gatekeeper” Phe-215, as well as the conserved catalytic

residues, Cys-164, His-303, and Asn-336. However, half of the

conserved active site residues are uniquely altered in C. micro-

carpa QNS. Thus, Thr-132, Ser-133, and the gatekeeper Phe-

265 in M. sativa CHS are simultaneously substituted with Met,

A structural similarity search was performed, using the Dali

program (28). The cavity volume and the active site entrance

area were calculated with the program CASTP. All crystal-

lographic figures were prepared with PyMOL (DeLano

Scientific).

Site-directed Mutagenesis—The plasmids expressing the

mutants of C. microcarpa QNS (Y197A) and C. microcarpa

ACS (S132M, T194M, and T197Y) were constructed with

a QuikChange site-directed mutagenesis kit (Stratagene),

according to the manufacturer’s protocol, using the following

pairs of primers (mutated codons are underlined): Y197A (5Ј-

GATATCATGAACATGGCTTTTCATGAGCCG-3Ј and 5Ј-

CGGCTCATGAAAAGCCATGTTCATGATATC-3Ј) for C.

microcarpa QNS, and S132M (5Ј-CATCTCATTTTCTGCAC-

AATGGCAGGCGTCGACATGCC-3Ј and 5Ј-GGCATGTCG-

ACGCCTGCCATTGTGCAGAAAATGAGATG-3Ј), T194M

(5Ј-GTTTGCTCTGAGAACATGATCCCCACTTTCCGT-

G-3Ј and 5Ј-CACGGAAAGTGGGGATCATGTTCTCAGA-

GCAAAC-3Ј), and T197Y (5Ј-GAGAACACAATCCCCTAT-

TTCCGTGGGCCG-3Ј and 5Ј-CGGCCCACGGAAATAGG-

GGATTGTGTTCTC-3Ј) for C. microcarpa ACS. The mutant

enzymes were expressed and purified with the same procedures

as described for the wild-type enzymes and used for the enzyme

reaction.

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FIGURE 3. Phylogenetic tree analysis of plant and bacterial type III PKSs. Multiple sequence alignment, performed with ClustalW (1.8). The scale represents

0.1 amino acid substitutions per site. Abbreviations used are as follows: ALS, aloesone synthase; BBS, bibenzyl synthase; BIS, biphenyl synthase; BPS, benzo-

phenone synthase; CTAS, 4-coumaroyltriacetic acid synthase; CUS, curcuminoid synthase; CURS, curcumin synthase; DCS, diketide-CoA synthase; HKS, hexa-

ketide synthase; OKS, octaketide synthase; ORAS, 3Ј-oxoresorcinolic acid synthase; PCS, pentaketide chromone synthase; 2PS, 2-pyrone synthase; STS, stilbene

synthase; THNS, tetrahydroxynaphthalene synthase; VPS, valerophenone synthase. The ␤-ketoacyl carrier protein synthase III (KAS III and FABH) enzymes of

E. coli were employed as out groups.

Ala, and Leu, respectively (Fig. 2). In addition, Met-137, Thr- and malonyl-CoA as substrates. Although the phylogenetic tree

194, Thr-197, Gly-256, and Ser-338 of M. sativa CHS are char- analyses predicted a close relationship between C. microcarpa

acteristically altered to Ile, Met, Tyr, Ala, and Gly, respectively. QNS and the biphenyl-producing biphenyl synthase (Fig. 3),

The G256A/S338G substitutions are also found in Sorbus aucu- the LC-ESI-MS analyses of the enzyme reaction products dem-

paria biphenyl synthase (30) and Hypericum androsaemum onstrated that it does not produce biphenyl from benzoyl-CoA

benzophenone synthase (17). The conserved active site resi- (Fig. 4C). Instead, C. microcarpa QNS efficiently accepts

dues Thr-197/Gly-256/Ser-338 are altered in a number of func- N-methylanthraniloyl-CoA as a starter substrate to produce

tionally different type III PKSs and are thought to be crucial for the diketide 4-hydroxy-N-methylquinolone as the single prod-

governing the substrate and product specificities of the enzyme uct by the condensation of one molecule of malonyl-CoA (Fig.

reactions (1, 2). However, in C. microcarpa ACS, Thr-132, Ser- 4A). Notably, C. microcarpa QNS did not accept 4-coumaroyl-

133, and Phe-265 are substituted with Ser, Ala, and Val, respec- CoA as a substrate (Fig. 4B) and produced only triketide lac-

tively, as in the case of R. graveolens ACS and A. marmelos QNS tones from benzoyl-CoA and hexanoyl-CoA as starters (Fig. 4,

(Fig. 2). These three residues are reportedly crucial for the sub- C and D). No additional products were detected in all of the

strate and product specificities of the enzyme reactions in A. enzyme reactions tested, even with changes in the reaction pH,

marmelos QNS and R. graveolens ACS (12, 15). A phylogenetic temperature, and time. The steady-state kinetics values for

tree analysis grouped C. microcarpa QNS and ACS with the quinolone formation by C. microcarpa QNS were K_mϭ 37.8

Ϫ1

non-chalcone-producing enzymes and the closely related ␮M, k_catϭ 15.7 min^Ϫ1, and k_cat/K_mϭ 416 min^Ϫ1mM for

biphenyl- and alkaloid-producing type III PKSs, respectively N-methylanthraniloyl-CoA, with a pH optimum at 8.0 within a

(Fig. 3).

range of 6.5–8.5. The steady-state kinetics values for quinolone

In Vitro Analysis of C. microcarpa QNS Activity—The formation were better than those of R. palmatum BAS (K_mϭ

sequence analyses suggested that both of the newly obtained C. 23.7 ␮M, k_catϭ 1.5 min^Ϫ1, k_cat/K_mϭ 62.4 min^Ϫ1mM^Ϫ1) (16) but

microcarpa type III PKSs are functionally distinct from the reg- not as good as those of A. marmelos QNS (K_mϭ 2.9 ␮M, k_catϭ

ular CHS and could possess interesting catalytic activities. The 3.8 min^Ϫ1, k_cat/K_mϭ 1290 min^Ϫ1mM^Ϫ1) (Table 1) (12).

recombinant C. microcarpa QNS was functionally expressed in

In Vitro Analysis of C. microcarpa ACS Activity—The phylo-

E. coli as an N-terminally His₆-tagged protein and was purified genetic tree analyses suggested that C. microcarpa ACS is

and subjected to enzyme reactions using N-methylanthra- closely related to the acridone-producing R. graveolens ACS

niloyl-CoA, 4-coumaroyl-CoA, benzoyl-CoA, hexanoyl-CoA, and A. marmelos QNS (Fig. 3). Indeed, C. microcarpa ACS effi-

28850 JOURNAL OF BIOLOGICAL CHEMISTRY

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Quinolone and Acridone Synthases from Citrus Microcarpa

FIGURE 4. HPLC elution profiles of the enzyme reaction products of C. microcarpa QNS and ACS. A–D, enzyme reaction products of C. microcarpa (Cm) ACS

and QNS from N-methylanthraniloyl-CoA (A), 4-coumaroyl-CoA (B), benzoyl-CoA (C), and hexanoyl-CoA, and malonyl-CoA (D). Note that by acid treatment

naringenin chalcone is converted to racemic naringenin (5,7,4Ј-trihydroxyflavanone) through a nonstereospecific ring-C closure.

TABLE 1

Steady-state kinetic parameters of C. microcarpa QNS and ACS

Product

K_m

␮M

k_cat

min

k

_cat/K_m

Ref.

Ϫ1

min^Ϫ1mM

N-methylanthraniloyl-CoA

C. microcarpa QNS

A. marmelos QNS

Quinolone

Acridone

37.8 Ϯ 3.2

2.9

15.7 Ϯ 2.0

3.8

416

1290

62.4

117

324

This paper

12

R. palmatum BAS

23.7

1.5

16

C. microcarpa ACS

37.4 Ϯ 2.4

4.3 Ϯ 1.7

4.0 Ϯ 0.6

1.4 Ϯ 0.0

This paper

4-Coumaroyl-CoA

C. microcarpa ACS

M. sativa CHS

Naringenin

13.6 Ϯ 3.1

5.1

6.8 Ϯ 2.2

6.1

500

843

45.5

179

This paper

13

Naringenin

C. microcarpa QNS Y197A

R. palmatum BAS

Benzalacetone

28.3 Ϯ 5.7

10.0

1.3 Ϯ 0.2

1.8

This paper

19

ciently accepted N-methylanthraniloyl-CoA as the starter sub- respect to the quinolone forming activity, the kinetics values

strate to produce the tetraketide 1,3-dihydroxy-N-methylacri- were K_mϭ 34.4 ␮M, k_catϭ 4.0 min^Ϫ1, and k_cat/K_mϭ 117

Ϫ1

done after sequential condensations with three molecules of min^Ϫ1mM for N-methylanthraniloyl-CoA, with a pH opti-

malonyl-CoA, along with 4-hydroxy-N-methylquinolone and mum at 8.0 within a range of 6.5–8.5 (Table 1). Thus, the cata-

N-methylanthraniloyltriacetic acid lactone (Fig. 4A). The lytic efficiency for the formation of the acridone is 2.8-fold

steady-state kinetics values for acridone formation by C. micro- higher than that of the quinolone. In addition, C. microcarpa

carpa ACS were K_mϭ 4.3 ␮M, k_catϭ 1.4 min^Ϫ1, and k_cat/K_mϭ ACS also accepted 4-coumaroyl-CoA as a starter substrate to

Ϫ1

324 min^Ϫ1mM

for N-methylanthraniloyl-CoA, with a pH yield naringenin chalcone, along with triketide and tetraketide

optimum at 8.0 within a range of 6.5–8.5. However, with lactone derailment by-products (Fig. 4B). When benzoyl-CoA

OCTOBER 4, 2013•VOLUME 288•NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 28851

Quinolone and Acridone Synthases from Citrus Microcarpa

TABLE 2

tively). A structure-based similarity search using the Dali pro-

Data collection and refinement statistics

gram revealed that the overall structures of M. sativa CHS,

Huperzia serrata PKS1, and R. palmatum BAS, which are func-

tionally related to C. microcarpa QNS and ACS, exhibited

r.m.s.d. of 0.9, 0.9, and 0.7 Å to that of C. microcarpa QNS, and

0.5, 0.7, and 0.6 Å to that of C. microcarpa ACS, respectively.

Active Site Structure of C. microcarpa QNS—Similar to C.

microcarpa ACS, the CHS’s conserved gatekeeper Phe-265 is

substituted with Leu in C. microcarpa QNS. The aromatic side

chain of the highly conserved Phe-265 in the plant type III PKSs

usually blocks the access of the residues behind it to the active

site cavity. In addition, Thr-132, Ser-133, Met-137, Thr-194,

Thr-197, Gly-256, and Ser-338 in M. sativa CHS are character-

istically replaced by Met, Ala, Ile, Met, Tyr, Ala, and

Gly, respectively. No such simultaneous substitutions were

observed in the primary structures of the other known type III

PKSs, suggesting that these changes control the unique sub-

strate and product specificities of C. microcarpa QNS. These

residues are spatial analogs among the structures of C. micro-

carpa QNS and M. sativa CHS. The F265L substitution in C.

microcarpa QNS not only grossly changes the shape of the

active site cavity, but also permits Leu-267, behind Leu-265, to

form part of the active site entrance (Fig. 6A). Furthermore,

Data collection

C. microcarpa QNS

C. microcarpa ACS

Unit cell parameter

Space group

a, b, c

P2₁

51.7, 135.9, 107.6 Å

P6₅22

106.0, 106.0, 346.5 Å

Resolution range 50 to 2.47 Å (2.51 to 2.47 Å) 50 to 2.35 Å (2.39 to 2.35 Å)

Completeness

͗I/␴I͘

99.4% (98.7%)

10.7% (2.10%)

10.3% (58.0%)

3.85 (3.85)

99.9% (100%)

60.9% (10.0%)

7.4% (36.1%)

21.0 (17.2)

R_merge

Redundancy

No. of observed

reflections

No. of unique

reflections

188,726

1,031,666

49,068

49,243

Refinement

Resolution

20.0 to 2.47 Å

19.5%

34.5 to 2.35 Å

18.3%

Overall R_work

Overall R_free

Total atoms

No. of protein

atoms

24.1%

21.8%

12238

6624

12028

6173

No. of waters

No. of ligand

Average B-factors

Protein atoms

Waters

198

12

331

120

39.1 Å²

33.3 Å²

49.6 Å²

33.2 Å²

33.3 Å²

56.2 Å²

Ligands

r.m.s.d. from ideal

Bond length

Bond angles

0.01 Å

1.301°

0.009 Å

1.208°

or hexanoyl-CoA was tested as the starter substrate, C. micro- because of the absence of the aromatic moiety, the side chain of

carpa ACS afforded the aromatic tetraketides 2,4,6-trihydroxy- Phe-215 protrudes more toward Leu-265, as compared with

benzophenone and 2-(1-keto-hexyl)phloroglucinol, but much that of Phe-215 in M. sativa CHS, thus widening part of the

less efficiently (Fig. 4, C and D). With respect to the chalcone active site entrance.

forming activity, C. microcarpa ACS showed K_mϭ 13.6 ␮M,

The structural comparison also revealed the significant con-

k

_catϭ 6.8 min^Ϫ1, and k_cat/K_mϭ 500 min^Ϫ1mM^Ϫ1for 4-couma- formational differences of residues 159–164 (r.m.s.d. of 1.1 Å

royl-CoA, and the latter value is 1.7-fold lower than that of M. for the C␣ atoms) and the slight displacement of an ␣-helix

sativa CHS (K_mϭ 5.1 ␮M, k_catϭ 6.1 min^Ϫ1, and k_cat/K_mϭ 843 consisting of residues 164–179 in C. microcarpa QNS. These

min^Ϫ1mM^Ϫ1) (13) (Table 1).

conformational changes are presumably caused by various

Overall Structures of C. microcarpa QNS and ACS—The apo- neighboring amino acid replacements in C. microcarpa QNS,

crystal structure of the recombinant C. microcarpa QNS and such as the large-to-small M159I and small-to-large A166I sub-

the crystal structure of the recombinant C. microcarpa ACS stitutions in these regions. As a result, the C␣ atom of the cat-

complexed with CoA-SH were solved at 2.47 and 2.35 Å reso- alytic residue Cys-164 in C. microcarpa QNS is displaced by 1.7

lution, respectively. The crystallographic data and refinement Å toward the outside of the CHS’s active site cavity. This alters

statistics are summarized in Table 2. The asymmetric units of the shape and size of the active site cavity and also widens the

C. microcarpa QNS and ACS contained four and two nearly active site entrance. The estimated total area of the active site

identical monomers, respectively, and significant backbone entrance of QNS is 47 Å², which is 2.8 and 1.5 times larger than

changes were not observed between the monomers in these those of M. sativa CHS (17 Å²) and C. microcarpa ACS (33 Å²)

structures. The structures of C. microcarpa QNS and ACS, (Fig. 6, A–C), respectively.

sharing 56% amino acid sequence identity, are nearly identical,

However, the side chain of Met-132 of C. microcarpa QNS,

with root-mean-square deviations (r.m.s.d.) of 0.9 Å. The over- corresponding to Thr-132 in M. sativa CHS, occupies the so-

all structures of C. microcarpa QNS and ACS revealed the con- called coumaroyl-binding pocket that accommodates the aro-

servation of the ␣␤␣␤␣-fold, observed in all structurally char- matic moiety of the coumaroyl starter in M. sativa CHS (Fig. 7,

acterized type III PKSs (Fig. 5). The catalytic triad consisting of A and C). The side chain of Met-194 in C. microcarpa QNS

Cys-164, His-303, and Asn-336 is buried deep within each protrudes toward Gly-338, as compared with CHS’s Thr-194.

monomer, at the intersection of a characteristic 16-Å-long CoA The coumaroyl-binding pocket in M. sativa CHS is thus absent

binding tunnel and a large cavity, in a location and an orienta- from the active site cavity of C. microcarpa QNS, along with

tion very similar to those of the other plant type III PKSs (6, slight differences in the various surrounding amino acids.

31–35). Ile-137 in C. microcarpa QNS and Met-137 in C. micro-

Interestingly, the side chain of Tyr-197 in C. microcarpa

carpa ACS, corresponding to Met-137 of M. sativa CHS, pro- QNS, corresponding to Thr-197 lining the bottom of the active

trude into the other monomer and form part of the active site site cavity of M. sativa CHS, is inserted in front of the side chain

wall, as in the case of M. sativa CHS. The overall structures of C. of Met-263, corresponding to Leu-263 in M. sativa CHS, and its

microcarpa QNS and ACS are highly homologous to those of terminal hydroxyl group forms a hydrogen bond with the sulfur

the structurally characterized plant type III PKSs (r.m.s.d. 0.7– atom of Met-132 (Fig. 7, A and C). The substitution of Thr-197

1.3 Å and 0.5–1.2 Å for C. microcarpa QNS and ACS, respec- with Tyr in C. microcarpa QNS thereby drastically narrows the

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Quinolone and Acridone Synthases from Citrus Microcarpa

FIGURE 5. Overall structures of C. microcarpa QNS and ACS. A, C. microcarpa QNS; B, C. microcarpa ACS. The structures are represented as schematics. The

catalytic Cys-164 and the substrate entrance are represented by a CPK model and an arrow, respectively. The CoASH molecules in C. microcarpa ACS are

depicted as blue stick models.

FIGURE 6. Comparison of the active site entrances of C. microcarpa QNS/ACS and other type III PKSs. A, C. microcarpa QNS; Bl C. microcarpa ACS, and C, M.

sativa CHS. Arrows indicate the substrate entrance in each structure.

active site cavity of C. microcarpa QNS, especially at the bot- pates in the formation of the active site entrance, with a slight

tom. Additionally, the side chain of Ala-256, which is the spatial displacement of its C␣ atom and a significant shift of its torsion

analog of Gly-256 of M. sativa CHS in C. microcarpa QNS, angle toward Val-265 (Fig. 6B). In addition, presumably

protrudes toward Tyr-197 and constricts the bottom part of the because of the loss of the aromatic side chain at this position

active site cavity of C. microcarpa QNS, along with the side and of several slight conformational differences between both

chain of Tyr-197. In contrast, the large-to-small S338G and enzymes, such as the G216A substitution in C. microcarpa

M137I substitutions in C. microcarpa QNS slightly expand its ACS, the backbone torsion angle of Phe-215 (Ϫ53, 136), as

active site wall near the active site entrance, as compared with compared with that of Phe-215 (Ϫ79, 138) in M. sativa CHS, is

M. sativa CHS. The total cavity volume (290 Å³) of the active shifted by a ␾ angle of Ϫ26° and a ␺ angle of ϩ2°. This is accom-

site of C. microcarpa QNS is about 2.6 times smaller than those panied by a slight displacement of its C␣ atom, and the side

of M. sativa CHS (750 Å³) and C. microcarpa ACS (760 Å³) (Fig. chain of Phe-215 protrudes toward Val-265 (Fig. 6B). These

7, A–C).

conformational differences expand the entrance to the active

Active Site Structure of C. microcarpa ACS—One of the char- site cavity of C. microcarpa ACS, as compared with that of M.

acteristic features of the C. microcarpa ACS sequence is the sativa CHS. As a result, the estimated total area of the C. micro-

characteristic substitution of the CHS’s conserved active site carpa ACS’s entrance is 33 Å², which is twice as large as that of

residues Thr-132, Ser-133, and Phe-265 with Ser, Ala, and Val, M. sativa CHS (17 Å²) (Fig. 6, B and C).

respectively. As in the case of C. microcarpa QNS, the location

In addition to these significant conformational changes, the

of Val-265 in the active site cavity of C. microcarpa ACS is side chains of Val-265 and Ser-132, as compared with those of

similar to that of Phe-265 in M. sativa CHS. The loss of the Phe-265 and Thr-132 in M. sativa CHS, slightly protrude

aromatic moiety at this position obviously changes the shape of toward the inside and the outside of the active site cavity of C.

the active site cavity of C. microcarpa ACS. Furthermore, the microcarpa ACS, respectively (Fig. 7, B and C). With the slight

side chain C␦1 of Leu-267, corresponding to Leu-267 behind displacements of their C␣ atoms and the backbone torsion

the aromatic side chain of Phe-265 in M. sativa CHS, partici- angles, these conformational changes contribute toward alter-

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Quinolone and Acridone Synthases from Citrus Microcarpa

FIGURE 7. Comparison of the active site structures of C. microcarpa QNS and ACS and other type III PKSs, and their schematic representations. A, C.

microcarpa QNS; B, C. microcarpa ACS; C, M. sativa CHS, and D, R. palmatum BAS. The naringenin in M. sativa CHS and the bound monoketide intermediate in R.

palmatum BAS are shown as magenta and pink stick models, respectively. Arrows indicate the substrate entrance in each structure.

ing the shape of the active site cavity of C. microcarpa ACS. mutagenesis and investigated the mechanistic consequences of

However, Ala-133 occupies almost the same position as the the point mutations. First, to test the hypothesis that the bulky

active site residue Ser-133 of M. sativa CHS, with an orientation Tyr-197 plays a crucial role in the active site architecture of C.

very similar to that of Ser-133. The only difference is the steric microcarpa QNS, we constructed the Y197A mutant of C.

bulk between the side chains of Ala and Ser at this position. No microcarpa QNS, in which the bulky Tyr-197 was substituted

other significant conformational differences were observed with a small Ala. As a result, the Y197A mutant produced the

between both structures. The estimated total cavity volume of diketide quinolone, from N-methylanthraniloyl-CoA, almost as

C. microcarpa ACS is 760 Å³, which is almost the same as that efficiently as the wild-type enzyme; however, interestingly, the

of M. sativa CHS (750 Å³) (Fig. 7, B and C).

QNS mutant now accepted 4-coumaroyl-CoA as the starter

Structure-based Mutagenesis of C. microcarpa QNS and ACS— substrate to produce the diketide benzalacetone and triketide

To further clarify the structure-function relationship of C. lactone (Fig. 8A). The kinetics values for benzalacetone forma-

Ϫ1

microcarpa QNS and ACS, we performed site-directed tion by the Y197A mutant were K_mϭ 28.3 ␮M, k_catϭ 1.3 min

,

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Quinolone and Acridone Synthases from Citrus Microcarpa

FIGURE 8. HPLC elution profiles of the enzyme reaction products of C. microcarpa QNS and ACS mutants. A, enzyme reaction products of C. microcarpa

QNS, wild-type, and Y197A mutant, from 4-coumaroyl-CoA. B and C, enzyme reaction products of C. microcarpa ACS, wild-type, and mutants (S132M, T194M,

andT197Y), fromN-methylanthraniloyl-CoA(B), and4-coumaroyl-CoA(C). Notethatbyacidtreatmentnaringeninchalconeisconvertedtoracemicnaringenin

(5,7,4Ј-trihydroxyflavanone) through a nonstereospecific ring-C closure.

and k_cat/K_mϭ 45.5 min^Ϫ1mM^Ϫ1. Thus, the catalytic efficiency exhibits quite unique substrate and product specificities. The

for the formation of the benzalacetone is 3.9-fold lower than enzyme did not accept 4-coumaroyl-CoA as a substrate and

that of the native R. palmatum BAS (K_mϭ 10.0 ␮M, k_catϭ 1.8

produced only triketide lactones from benzoyl-CoA and hexa-

noyl-CoA as starter substrates (Fig. 1, B–D). These results sug-

gested that C. microcarpa QNS can be regarded as a dedicated

quinolone-producing enzyme. In contrast, the previously

reported A. marmelos QNS did not produce the quinolone

specifically, because it also generated the tetraketide 1,3-

dihydroxy-N-methylacridone from N-methylanthraniloyl-

CoA (12). Thus, A. marmelos QNS could be regarded as

another acridone-producing ACS, which simply yields the

diketide quinolone as a by-product. This is analogous to the

tetraketide chalcone-forming CHS, which also produced

triketide and tetraketide lactone by-products in the in vitro

enzyme reactions. Furthermore, another major difference is

that the quinolone- and acridone-forming A. marmelos QNS

also accepted 4-coumaroyl-CoA to produce the diketide ben-

zalacetone (12). However, we previously reported that the

diketide-forming R. palmatum BAS also accepted both 4-cou-

min^Ϫ1, k_cat/K_mϭ 179 min^Ϫ1mM^Ϫ1) (Table 1).

However, the crucial active site residues, Ser-132, Thr-194,

and Thr-197 in C. microcarpa ACS, were replaced with Met,

Met, and Tyr, respectively, as in the case of C. microcarpa QNS.

Notably, consistent with our hypothesis, all the point mutants

efficiently produced quinolone as the single product and lost

the tetraketide acridone and lactone producing activities

from the N-methylanthraniloyl-CoA starter (Fig. 8B). Fur-

thermore, as in the case of C. microcarpa QNS, all the ACS

mutants no longer accepted 4-coumaroyl-CoA as the starter

substrate to produce chalcone, tetraketide, and triketide lac-

tones (Fig. 8C).

DISCUSSION

In vitro assays of the enzymes clearly demonstrated that C.

microcarpa QNS is a novel type III PKS that produces the

diketide 4-hydroxy-N-methylquinolone as the single product maroyl-CoA and N-methylanthraniloyl-CoA as starter sub-

by the one-step condensation of N-methylanthraniloyl-CoA strates to produce the benzalacetone and quinolone scaffolds,

with malonyl-CoA (Fig. 1A). Notably, C. microcarpa QNS respectively (16, 19). These observations indicated that the sub-

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Quinolone and Acridone Synthases from Citrus Microcarpa

strate preferences of C. microcarpa QNS, A. marmelos QNS,

and R. palmatum BAS are significantly different, due to the

modifications of their active site architectures, as discussed

below.

The crystal structure of C. microcarpa ACS also revealed a

wide active site entrance (37 Å²), as in the case of C. microcarpa

QNS (47 Å²) (Fig. 6B). However, unlike C. microcarpa QNS, the

broadening of the active site entrance of C. microcarpa ACS is

caused by the F265V substitution and the conformational

changes of Phe-215 and Leu-267 (Fig. 6B). Furthermore, the

cavity volume of C. microcarpa ACS (760 Å³) is 2.6 times larger

than that of C. microcarpa QNS (290 Å³) and almost as large as

that of M. sativa CHS (750 Å³). The wide active site entrance

and the large active site cavity clearly affect the starter substrate

preference and the product specificity of C. microcarpa ACS.

As a result, C. microcarpa ACS accepts the bulky N-methylan-

thraniloyl-CoA as the starter substrate and catalyzes the itera-

tive condensations with three molecules of malonyl-CoA to

produce the acridone scaffold, by employing an active site cav-

ity and catalytic machinery similar to those of CHS. Further-

more, as in the cases of the other type III PKSs, C. microcarpa

ACS also exhibits promiscuous substrate specificity and

accepts various starter substrates to produce distinct molecular

scaffolds, including acridone, quinolone, chalcone, benzophe-

none, and phloroglucinol (Fig. 1, A–D). The site-directed

mutagenesis studies suggested that three active site residues

Ser-132, Thr-194, and Thr-197 play a crucial role for the

enzyme activity of C. microcarpa ACS. The S132M, T194M,

and T197Y point mutations resulted in the loss of the tetra-

ketide acridone and chalcone forming activities and production

of the diketide quinolone as the single product. Thus, C. micro-

carpa ACS was functionally converted into QNS. These results

clearly supported our hypothesis that the volume and shape of

the active site cavity control the product specificity of these

enzyme reactions.

Interestingly, the crystal structure revealed that C. micro-

carpa QNS has an unusually wide active site entrance (Fig. 6A).

Similar expansions of the active site entrances have also been

reported for the structures of the R. palmatum BAS (34) and

acridone-producing M. sativa F215S CHS mutant (13), which

both accept the bulky substrate N-methylanthraniloyl-CoA.

The active site entrance of C. microcarpa QNS (47 Å²) is larger

than that of R. palmatum BAS (34 Å²) and is almost as large

as that of the M. sativa F215S CHS mutant (41 Å²). In the

structure of C. microcarpa QNS, the broadening of the active

site entrance is caused by the F265L substitution and the devi-

ation of the catalytic residue Cys-164 toward the outside of the

active site cavity, as compared with the other type III PKSs.

However, in R. palmatum BAS, the F208L substitution and its

conformational changes expand the active site entrance. The

wide active site entrance thus provides enough space to facili-

tate the access of the bulky N-methylanthraniloyl-CoA starter

to the catalytic center of the enzymes.

In addition, the active site cavity volume of C. microcarpa

QNS is quite small (290 Å³) and is even smaller than that of the

diketide-forming R. palmatum BAS (350 Å³) (Fig. 7, A and D)

(34). Furthermore, the crystal structure revealed the absence of

the CHS’s coumaroyl-binding pocket in the active site of C.

microcarpa QNS, due to the unique substitutions of CHS’s con-

served Thr-132, Thr-194, and Thr-197 with Met, Met, and Tyr,

respectively. This is the reason why C. microcarpa QNS does

not accept 4-coumaroyl-CoA as a substrate. In contrast,

although R. palmatum BAS also lacks the coumaroyl-binding

pocket, this enzyme utilizes novel alternative pockets to bind

the coumaroyl starter and produce the diketide benzalacetone

(Fig. 7D) (34). Thus, the shape and the cavity volume of C.

microcarpa QNS restrict the binding of the coumaroyl starter

and the malonyl-CoA extender. Indeed, the mutagenesis anal-

yses revealed that the bulky Tyr-197 drastically narrows down

the active site cavity of C. microcarpa QNS and thereby con-

trols the product chain length and specificity of the enzyme

reaction. It is remarkable that the large-to-small Y197A mutant

of C. microcarpa QNS gained the function of benzalacetone

and triketide lactone-producing activities by the single amino

acid substitution. Notably, similar steric contraction of the

active site cavity was also observed for Gerbera hybrida 2-meth-

ylpyrone synthase, which utilizes the small acetyl-CoA as a

starter substrate to produce triacetic acid lactone (31). The

active site cavity of G. hybrida 2-pyrone synthase is even

smaller (250 Å³) and also lacks the coumaroyl-binding pocket

(36, 37). These observations support the unique catalytic activ-

ity of C. microcarpa QNS, which accepts the N-methylanthra-

niloyl-CoA starter through its wide active site entrance and

catalyzes the condensation with malonyl-CoA. The chain elon-

gation reaction is terminated at the diketide stage due to the

steric contraction of the active site cavity, and this is followed by

the N/C1 intramolecular lactamization of the Cys-bound linear

diketide intermediate and concomitant thioester bond cleavage

to produce the quinolone scaffold.

In the generation of the acridone tricyclic ring system, one of

the intriguing points is the timing of the C–N bond formation,

and the C-ring-forming reaction involving Claisen-type C–C

bond formation and concomitant thioester bond cleavage of

the linear tetraketide intermediate from the active site Cys.

Interestingly, previously reported experiments for the forma-

tion of rutacridone with [1-C¹³]-, [2-C¹³]-, and [1,2-C¹³]acetate

revealed two different labeling patterns within the C-ring (38).

This result indicated that the C-ring is indeed derived from

three acetate units and suggested that the different labeling

patterns were caused by the rotation of a bicyclic N-methylami-

nobenzophenone intermediate (or a nonaromatized anthra-

niloyl-trione intermediate) prior to C–N bond formation. In

fact, many bicyclic N-methylaminobenzophenones have been

isolated from various Rutaceae plants (39–42). Our docking

simulations based on the crystal structure of C. microcarpa

ACS predicted that the active site cavity is large enough to

accommodate the linear or cyclized tetraketide intermediate;

however, there is not enough space for the rotation. The dock-

ing simulation with the linear tetraketide intermediate also pre-

dicted that the C1 carbon resides near the C6 carbon in the

active site cavity. Therefore, we propose that the formation of

the acridone tricyclic ring system proceeds by the initial cycli-

zation of the linear tetraketide intermediate by Claisen-type

condensation to produce the bicyclic N-methylaminobenzo-

phenone (or a nonaromatized anthraniloyltrione), which is fol-

28856 JOURNAL OF BIOLOGICAL CHEMISTRY

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Quinolone and Acridone Synthases from Citrus Microcarpa

Sugio, S., Kohno, T., and Abe, I. (2011) Synthesis of unnatural novel alka-

lowed by the C–N bond formation to generate the acridone

scaffold.

Finally, despite the high sequence identity with C. micro-

loid scaffolds by exploiting plant polyketide synthase. Proc. Natl. Acad. Sci.

U.S.A. 108, 13504–13509

7. Hayashi, H., Miwa, Y., Ichikawa, S., Yoda, N., Miki, I., Ishii, A., Kono, M.,

Yasuzawa, T., and Suzuki, F. (1993) 5-HT₃receptor antagonists. 2,4-Hy-

droxy-3-quinolinecarboxylic acid derivatives. J. Med. Chem. 36, 617–626

8. Dittmer, D. C., Li, Q., and Avilov, D. V. (2005) Synthesis of coumarins,

4-hydroxycoumarins, and 4-hydroxyquinolinones by tellurium-triggered

cyclizations. J. Org. Chem. 70, 4682–4686

carpa ACS and R. graveolens ACS, both accepting 4-couma-

royl-CoA as the starter substrate to produce chalcone, the pre-

viously reported acridone/quinolone-producing A. marmelos

QNS does not produce chalcone from 4-coumaroyl-CoA,

although these three enzymes share the simultaneous substitu-

tions of the CHS’s conserved active site residues Thr-132, Ser-

133, and Phe-265 with Ser, Ala, and Val, respectively. Site-di-

rected mutagenesis studies of R. graveolens ACS and A.

marmelos QNS indicated that the three residues play important

roles in the substrate and product specificities of the two

enzymes (12, 15). For example, the S132T/A133S double

mutant of A. marmelos QNS produced chalcone from 4-cou-

maroyl-CoA, whereas the S132T/A133S/V265F triple mutant

lost the enzyme activity, and no longer accepted the coumaroyl

starter to yield any products (12). However, the S132T/A133S/

V265F triple mutation of R. graveolens ACS caused full conver-

sion into a functionally distinct chalcone-forming enzyme (15).

These observations suggested that subtle structural differences

exist in the active site architectures of these three acridone-

producing enzymes.

9. Kelly, J. X., Smilkstein, M. J., Brun, R., Wittlin, S., Cooper, R. A., Lane, K. D.,

Janowsky, A., Johnson, R. A., Dodean, R. A., Winter, R., Hinrichs, D. J., and

Riscoe, M. K. (2009) Discovery of dual function acridones as a new anti-

malarial chemotype. Nature 459, 270–273

10. Lukacin, R., Springob, K., Urbanke, C., Ernwein, C., Schröder, G.,

Schröder, J., and Matern, U. (1999) Native acridone synthases I and II from

Ruta graveolens L. form homodimers. FEBS Lett. 448, 135–140

11. Springob, K., Lukacin, R., Ernwein, C., Gröning, I., and Matern, U. (2000)

Specificities of functionally expressed chalcone and acridone synthases

from Ruta graveolens. Eur. J. Biochem. 267, 6552–6559

12. Resmi, M. S., Verma, P., Gokhale, R. S., and Soniya, E. V. (2013) Identifi-

cation and characterization of a type III polyketide synthase involved in

quinolone alkaloid biosynthesis from Aegle marmelos correa. J. Biol.

Chem. 288, 7271–7281

13. Jez, J. M., Bowman, M. E., and Noel, J. P. (2002) Expanding the biosynthetic

repertoire of plant type III polyketide synthases by altering starter mole-

cule specificity. Proc. Natl. Acad. Sci. U.S.A. 99, 5323–5324

14. Lukacin, R., Schreiner, S., Silber, K., and Matern, U. (2005) Starter sub-

strate specificities of wild-type and mutant polyketide synthases from Ru-

taceae. Phytochemistry 66, 277–284

In conclusion, C. microcarpa QNS is a novel type III PKS that

produces the diketide quinolone by the one-step condensation

of N-methylanthraniloyl-CoA and malonyl-CoA, whereas C.

microcarpa ACS is a multifunctional PKS that produces not

only acridone, but also chalcone, benzophenone, and phloro-

glucinol. The x-ray crystal structures of C. microcarpa QNS and

ACS revealed the wide active site entrances of both enzymes,

which facilitate the binding of the bulky N-methylanthraniloyl-

CoA starter. The active site cavity of C. microcarpa QNS is

significantly smaller than that of ACS, which leads to the spe-

cific production of the quinolone scaffold, whereas C. micro-

carpa ACS utilizes a large cavity to yield the tetraketide acri-

done, by employing an active site cavity and catalytic machinery

similar to those of CHS. These results have provided the first

structural bases for the production of the anthranilate-derived

quinolone and acridone alkaloids by the type III PKSs. These

findings will enable further engineering of the enzymes to cre-

ate novel, structurally distinct, and biologically active molecu-

lar scaffolds for drug discovery.

15. Lukacin, R., Schreiner, S., and Matern, U. (2001) Transformation of acri-

done synthase to chalcone synthase. FEBS Lett. 508, 413–417

16. Abe, I., Abe, T., Wanibuchi, K., and Noguchi, H. (2006) Enzymatic forma-

tion of quinolone alkaloids by a plant type III polyketide synthase. Org.

Lett. 8, 6063–6065

17. Liu, B., Falkenstein-Paul, H., Schmidt, W., and Beerhues, L. (2003) Benzo-

phenone synthase and chalcone synthase from Hypericum androsaemum

cell cultures: cDNA cloning, functional expression, and site-directed mu-

tagenesis of two polyketide synthases. Plant J. 34, 847–855

18. Abe, I., Morita, H., Nomura, A., and Noguchi, H. (2000) Substrate speci-

ficity of chalcone synthase: enzymatic formation of unnatural polyketides

from synthetic cinnamoyl-CoA analogues. J. Am. Chem. Soc. 122,

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19. Abe, I., Takahashi, Y., Morita, H., and Noguchi, H. (2001) Benzalacetone

synthase. A novel polyketide synthase that plays a crucial role in the bio-

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VOLUME 288•NUMBER 40•OCTOBER 4, 2013

Enzymology:

Cloning and Structure-Function Analyses

of Quinolone- and Acridone-producing

Novel Type III Polyketide Synthases from

Citrus microcarpa

Takahiro Mori, Yoshihiko Shimokawa,