www.elsevier.com/locate/plantsci

Plant Science 171 (2006) 194–205

Screening for inhibitors of 2-oxoglutarate-dependent dioxygenases:

Flavanone 3b-hydroxylase and flavonol synthase§

Heidrun Halbwirth a, Thilo C. Fischer b, Karin Schlangen a, Wilhelm Rademacher c,Klaus-Jurgen Schleifer d, Gert Forkmann b, Karl Stich a,*

a Institut fur Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, Technische Universitat Wien, Getreidemarkt 9/1665, A-1060 Wien, Austriab Lehrstuhl fur Zierpflanzenbau, Technische Universitat Munchen Weihenstephan, Am Hochanger 4, D-85350 Freising, Germany

c BASF Aktiengesellschaft, Agrarzentrum, D-67114 Limburgerhof, Germanyd BASF Aktiengesellschaft, Forschung Wirk und Effektstoffe, D-67056 Ludwigshafen, Germany

Received 17 November 2005; received in revised form 12 March 2006; accepted 22 March 2006

Available online 18 April 2006

Abstract

2-Oxoglutarate-dependent dioxygenases (2-ODDs) catalyze numerous steps in biosynthetic pathways of plants. Prohexadione-Ca is a known

inhibitor of such reactions, due to its structural similarity to 2-oxoglutarate. In apple (Malus domestica) and pear (Pyrus communis) leaves, the

transient inhibition of 2-ODDs flavanone 3b-hydroxylase (FHT) and flavonol synthase (FLS) by prohexadione-Ca results in distinct changes in the

flavonoid spectrum, which are responsible for an enhanced resistance against two major pome fruit diseases, fire blight (caused by Erwinia

amylovora) and apple scab (caused by Venturia inaequalis). We used recombinant apple and pear FHT and apple FLS for screening 23 structural

analogues of 2-oxoglutarate, mostly cyclohexanediones, pyridine dicarboxylic acids and N-heterocycles with carbonyl functions for other

dioxygenase inhibitors. Activations, which were also observed for some compounds, are interpreted as in vitro effects due to Fe2+-chelating ability.

Apart from structural similarity to 2-oxoglutarate, close structural similarity of cyclohexanediones and some pyridine dicarboxylic acids to

flavonoid substrates was identified. Beyond the competitive inhibition for the co-substrate 2-oxoglutarate, flavonoid converting 2-ODDs may also

be inhibited at the substrate binding site by these inhibitors. All compounds found to be active as inhibitors may prove useful for studying the

reaction mechanisms and substrate specificities of various 2-ODDs.

# 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Pome fruits (apple, pear); Dioxygenase; Flavanone 3b-hydroxylase (FHT = F3H, EC 1.14.11.9); Flavonol synthase (FLS, EC 1.14.11.23);

Prohexadione-Ca; 2-Oxoglutarate analogous enzyme inhibitors

1. Introduction

Three enzymes of the flavonoid pathway in apple and pear

leaves are 2-oxoglutarate-dependent dioxygenases (2-ODDs):

flavanone 3b-hydroxylase (FHT, also F3H, EC 1.14.11.9),

flavonol synthase (FLS, EC 1.14.11.23), and anthocyanidin

Abbreviations: ANS, anthocyanidin synthase; DHK, dihydrokaempferol;

DHQ, dihydroquercetin; EGME, ethyleneglycol monomethylether; ERI, erio-

dictyol; FHT, flavanone 3-hydroxylase; FLS, flavonol synthase; NAR, narin-

genin; 2-ODD, 2-oxoglutarate-dependent dioxygenase§ The nucleotide sequences reported in this paper have been submitted to

GenBank under the accession numbers AY965339 (FHT Malus domestica cv.

Weirouge), AY965340 (FHT M. domestica cv. M9), AY965341 (FHT Pyrus

communis cv. Pyrodwarf), AY965342 (FHT P. communis cv. Conference) and

AY965343 (FLS M. domestica cv. M9).

* Corresponding author. Tel.: +43 1 58801 17320; fax: +43 1 58801 17399.

E-mail address: kstich@mail.zserv.tuwien.ac.at (K. Stich).

0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.plantsci.2006.03.014

synthase (ANS, EC 1.14.11.19) (Fig. 1). The acylcyclohex-

anedione prohexadione-Ca (CH1 in Fig. 2) – originally

developed as a plant growth retardant – is a structural analogue

of 2-oxoglutarate which competitively inhibits biosynthetic

steps catalyzed by 2-ODDs [1]. Several dioxygenases from

different pathways are affected. The inhibition of late steps of

gibberellin biosynthesis, leading to plants with a more compact

shoot, is of practical relevance in crop production [1]. Since

prohexadione-Ca is relatively short-lived and degrades into

natural compounds, it has favourable toxicological and

ecotoxicological features [1]. The transient inhibition of 2-

ODDs of the flavonoid pathway by prohexadione-Ca is of

interest, since it leads to significant changes in the flavonoid

spectrum of apple (Malus domestica) and pear (Pyrus

communis) leaves (Fig. 3). In particular, FHT inhibition leads

to the accumulation of its substrate eriodictyol, which is

subsequently converted to the 3-deoxyleucoanthocyanidin

H. Halbwirth et al. / Plant Science 171 (2006) 194–205 195

Fig. 1. Reactions catalyzed by FHT (A), FLS (B) and ANS (C).

luteoforol by the FNR side activity of the DFR enzyme.

Luteoforol is responsible for the enhanced resistance against

the serious pome fruit diseases fire blight and scab [2–6]. This

inhibition effect is not restricted to apple and pear, all FHT

enzymes tested so far from several other plant species are also

blocked by this compound [3,4,7].

In this study, 23 candidate compounds (Figs. 2 and 4) were

tested for their effects on recombinant FHT and FLS cloned

from apple and pear cultivars. The screening identified a

number of compounds with strong and specific influence on

FHT and FLS enzyme activities. Structure comparisons

indicate a parallel competition at the substrate binding site

of flavonoid processing 2-ODDs.

2. Material and methods

2.1. Plant materials

Young leaves of M. domestica cultivars ‘M9’ and ‘Weirouge’

and of P. communis cultivars ‘Pyrodwarf’ and ‘Conference’ were

supplied by D. Treutter (Technical University of Munich,

Freising-Weihenstephan, Germany). The leaves were harvested

during spring and summer 2000, frozen immediately with liquid

nitrogen and stored at �80 8C until use.

2.2. Chemicals

[2-14C]-Malonyl-coenzyme A (2 GBq/mmol) was obtained

from Amersham International (Freiburg, Germany). [14C]-

Naringenin (NAR) (0.8 GBq/mmol) was prepared as described

[8] using recombinant chalcone synthase in E. coli (J.

Schroeder, University of Freiburg, Germany) and chalcone

isomerase extracted from Dahlia flowers. [14C]-Eriodictyol

(ERI) (0.8 GBq/mmol) was synthesized from [14C]-naringenin

using microsomal preparations from Tagetes with high

flavonoid 30-hydroxylase activity [9]. [14C]-Dihydrokaemp-

ferol (DHK) (0.8 GBq/mmol) was synthesized as described [5].

Compounds NCO7–10, NCO12, and ACO were purchased

from Sigma–Aldrich Chemie GmbH (Taufkirchen, Germany),

NCO1–NCO15 and NCO11 from VWR International GmbH

(Darmstadt, Germany) and NCO6 from Acros Organics BVBA

(Geel, Belgium). CH1, CH2, CH8, CH9 and Daz were from

H. Halbwirth et al. / Plant Science 171 (2006) 194–205196

Fig. 2. Chemical structures of cyclohexanediones (in order to improve clarity, the nomenclature used for CH1–CH9 has been made uniform with regard to numbering

of the carbon atoms and, therefore, does not always comply with IUPAC or CAS rules) and some related compounds tested as potential dioxygenase inhibitors. The

structures of the dioxygenase cofactors 2-oxoglutarate and ascorbic acid are shown for comparison. CH1, calcium 3-hydroxy-5-oxo-4-propionyl-cyclohex-3-ene-1-

carboxylate (prohexadione-Ca); CH2, 3-hydroxy-5-oxo-4-cyclopropanecarbonyl-cyclohex-3-ene-1-carboxylic acid ethyl ester (trinexapac-ethyl); CH3: 3-hydroxy-

5-oxo-4-propionyl-cyclohex-3-ene-1-carbothioic acid S-ethyl ester; CH4, 3-hydroxy-5-oxo-4-propionyl-cylohex-3-ene-1-carbaldehyde; CH5, 3-hydroxy-5-oxo-4-

butyryl-cyclohex-3-ene-1-carboxylic acid ethyl ester; CH6, 3-hydroxy-5-oxo-4-propionyl-clohex-3-ene-1-(2-dimethylamino)-thiazole; CH7, 3-hydroxy-5-oxo-4-

propionyl-cyclohex-3-ene-1-pentanoic acid; CH8, sodium 4,6-dioxo-2,2-dimethyl-5-(1-alloxyamino-butylidene)-cyclohexane-1-carboxylic acid methyl ester

(alloxydim-Na); CH9, 3-hydroxy-5-oxo-4-(1-ethoxyimino-butyl)-cyclohex-3-ene-1-(tetrahydro-thiopyran-3-yl) (cycloxydim); ACO, benzene-1,2,4,5-tetracar-

boxylic acid; Daz: succinic acid 2,2-dimethyl hydrazide (daminozide).

H. Halbwirth et al. / Plant Science 171 (2006) 194–205 197

Fig. 3. The flavonoid biosynthesis in apple and pear and the influence of prohexadione-Ca.

LGC Promochem GmbH (Wesel, Germany). The acylcyclo-

hexanediones CH3–CH7 were synthesized at BASF Main

Laboratory (Ludwigshafen, Germany), following standard

procedures. Technical details are given in [10–12].

2.3. PCR amplification of FHT and FLS cDNAs

mRNA was isolated from leaf material of M. domestica

and P. communis cultivars using the mMACS mRNA Isolation

Kit1 (Miltenyi Biotec, 752-01). Reverse transcription was

performed with the SuperScript II1 Reverse Transcriptase

(Gibco BRL, 18064) and an anchored oligo-dT-primer. For

RT-PCR primers were constructed for the respective

untranslated 50- and 30-regions. For Malus FHT sequences

were available [13,14] and the primers GAA CCA ACA AAT

TCG ACA CC and GGA AGG AAA AGG TAC AAG TAG C

were used for the amplification of FHT from M. domestica cv.

Weirouge and cv. M9. For the Pyrus FHT several combina-

tions of primers constructed from Malus FHT sequences were

tested and selected for the amplification of fragments of the

expected size range. Amplification was possible with the

primers GAA CCA ACA AAT TCG ACA CC (both Pyrus

cultivars, untranslated 50-regions) and CCA CAA AGA GCT

TTC AAG TGA CTG G (P. communis cv. ‘Pyrodwarf’,

untranslated 30-region) or GGA AGG AAA AGG TAC AAG

TAG C (P. communis cv. ‘Conference’, untranslated 30-region). For the Malus FLS RT-PCR the primers CGA ACG

TCC AAA GCC CTT C and CCT GGA ACA CAA ATT CAA

ACA C were used relying on an available sequence [13]. All

amplifications were done with the Taq/Pwo-Polymerase

system ‘Expand High Fidelity PCR System1 (Roche,

1732641).

2.4. Cloning and heterologous expression in yeast

Cloning and heterologous expression of M. domestica cv.

M9 FHT and FLS and P. communis cv. Pyrodwarf FHT was

carried out using the commercially available yeast expression

system pYES2.1 (TOPO TA Cloning1 Kit, Invitrogen K4150-

01). All constructs were transformed into the yeast strain

InvSc1 (Invitrogen V825-20) using the S.c. Easy CompTM

Transformation Kit (Invitrogen K5050-01). The enzymes were

prepared from galactose-induced yeast cultures as described by

Urban et al. [15]. Transformation of yeast and preparation of

yeast-derived enzymes was also done with empty pYES2-

vector to provide a negative control for enzyme activities.

Protein content was determined by a modified Lowry procedure

[16] using BSA as a standard.

2.5. Standard enzyme assay

In a final volume of 100 ml the reaction mixture contained

10 ml enzyme preparation (17 mg total protein, recombinant

FHT preparation; 69 mg total protein, recombinant FLS

preparation), 80 ml 0.1 M K2HPO4/KH2PO4 (0.6 M sorbitol,

0.4% Na-ascorbate, pH 7.25), 108 Bq [14C]-labeled substrate

(0.148 nmol NAR for FHT, 0.095 nmol DHK for FLS),

50 nmol 2-oxoglutarate (in 5 ml distilled water) and was

started by the addition of 10 nmol FeSO4�7H2O (in 5 ml

distilled water). After 15 min (FHT) or 30 min (FLS) at 30 8C

H. Halbwirth et al. / Plant Science 171 (2006) 194–205198

Fig. 4. N-Heterocycles tested as potential dioxygenase inhibitors. NCO1, pyridine-2,4-dicarboxylic acid; NCO2, pyridine-2,5-dicarboxylic acid; NCO3, pyridine-

2,6-dicarboxylic acid; NCO4, pyridine-3,4-dicarboxylic acid; NCO5, pyridine-3,5-dicarboxylic acid; NCO6, pyridine-2,6-dicarboxylic acid chloride; NCO7,

pyridine-2,3-dicarboxylic acid (quinolinic acid); NCO8, pyrrole-3,4-dicarboxylic acid diethyl ester; NCO9, pyrazole-3,5-dicarboxylic acid (hydrate); NCO10,

piperidine-2-carboxylic acid; NCO11, piperazine-1,4-bis-(2-ethane sulphonic acid); NCO12, piperidine-4-carboxylic acid.

the reaction was stopped with 10 ml glacial acetic acid. The

phenolic compounds were extracted with 70 ml EtOAc, the

organic phases were applied to a precoated cellulose plate

(1.0571.001, Merck, Darmstadt, Germany) and chromato-

graphed with chloroform/acetic acid/water (10:9:1) as a solvent

system. Heterologous expression of Malus DFR in yeast and

enzymatic reactions were performed as previously described

[5], but using the same buffer as required for the FHT and FLS

assays. Radioactivity was detected and quantified using a

Typhoon 8600 Imager (Amersham). Recovery of [14C]-labeled

substrates was 95%. HPLC analysis was performed as

described [17] using a Perkin-Elmer Series 200 system

equipped with a UV detector coupled with a 500TR Flow

Scintillation Analyzer for the detection of radiolabeled

substances.

2.6. Enzyme characterization

Determination of the pH-optimum was carried out as

described for the standard assay, but using 0.2 M KH2PO4–

K2HPO4 buffer (containing 0.4% sodium-ascorbate) with pH

values between 6.0 and 8.5. Dependence of the enzymatic

turnover on time was determined during a period of 60 min at

intervals of 5 min. Kinetic data were calculated from Line-

weaver–Burk plots. All data represent an average of at least

three independent experiments.

2.7. Inhibitor studies

FHTassays were performed with NAR, FLS and DFR assays

with DHK as substrates. The screening was performed with two

final inhibitor concentrations of 1 and 0.1 mM. All inhibitor

solutions were freshly prepared. NCO1, NCO8–10, ACO, CH2,

CH3, CH5–CH9 and Daz were dissolved in methanol, NCO2,

NCO4 and NCO7 in DMSO, and NCO3, NCO6 and CH4 in

EGME. NCO12 was dissolved in water and CH1 in 0.05 M

McIlvaine buffer (pH 4). NCO5 and NCO11 were dissolved in

0.05 M Tris/HCl (pH 8.5). For methanol soluble inhibitors,

30 ml (for FLS and FHT assays) or 15 ml (for DFR assays) of

the 3.33 mM solutions were transferred into 1.5 ml reaction

H. Halbwirth et al. / Plant Science 171 (2006) 194–205 199

tubes together with the substrates and the solvent was removed

by vacuum. For the other inhibitors, 5 ml (for FLS and FHT

assays) or 2.5 ml (for DFR assays) of 20 mM stock solutions

were transferred directly into the reaction tubes before starting

the assay. In these cases, the control also contained the

respective solvent. Determination of IC50 values was carried

out according to [18]. All data represent an average of at least

three independent experiments.

2.8. Structural comparisons

Three-dimensional structure superimpositions were carried

out using the software package SYBYL1 7.0, Tripos Inc., St.

Louis, MO, USA.

3. Results and discussion

FHT and FLS were chosen as two major targets of

prohexadione-Ca. We isolated the cDNAs of FHT from apple

and pear leaves as well as FLS from apple leaves, and used the

recombinant enzymes to discover other inhibitors of dioxy-

genases or of specific flavonoid enzymes.

Fig. 5. Alignment of FHT genes of Malus domestica (FHT-M) and Pyrus communis

(Lee et al., GenBank); M. sp., Malus species (Davies [14]); Wr, Weirouge; M9, cv.

homologous. Some exchanges seem to be specific for the genera.

3.1. Cloning and heterologous expression

The FHT-cDNA clones of the Malus cultivars ‘M9’ and

‘Weirouge’ were obtained via RT-PCR, based on the

sequence information present in the databases [13,14]. Both

cDNAs showed few exchanges on the amino acid level

(Fig. 5) and consequently only M9 FHT was used for

heterologous expression and inhibitor screening. No sequence

information was available for Pyrus, therefore, the isolation

of the respective cDNA clones was performed by using

different primer combinations derived from the untranslated

50- and 30- sequences of Malus FHT for RT-PCR. By this

approach the FHT cDNAs of the Pyrus cultivars ‘Pyrodwarf’

and ‘Conference’ were obtained and found to be identical at

amino acid sequence (Fig. 5). The Pyrus cDNAs also turned

out to be closely homologous to the Malus cDNAs, some

exchanges are obviously genera specific. The FLS cDNA of

the M. domestica cultivar ’M9’ was also cloned by RT-PCR

using the available sequence information from Fuji apple

[13]. There was one (A108G) amino acid exchange between

the cDNA of the M. domestica cultivar ‘Fuji’ and the M.

domestica rootstock ‘M9’.

(FHT-P) cultivars. Variable positions are shown in bold print. Cultivars: Fj, Fuji

M9; Pd, Pyrodwarf; Co, Conference. The amino acid sequences are all closely

H. Halbwirth et al. / Plant Science 171 (2006) 194–205200

3.2. Verification of the recombinant enzymes and

optimization of the enzyme assays

Final proofs that the isolated cDNA clones from Malus cv.

‘M9’ and Pyrus cv. ‘Conference’ encode FHT or FLS were

achieved by assays with the enzymes functionally expressed in

yeast. [14C]-Products were identified by HPLC co-chromato-

graphy with authentic reference substances (Rt DHK:

21.40 min; Rt DHQ: 19.94 min; Rt kaempferol: 26.93 min; Rt

quercetin: 25.23 min).

3.2.1. Flavanone 3b-hydroxylase (FHT)

Incubation of the flavanones [14C]-NAR or [14C]-ERI with

enzyme preparations of recombinant Malus and Pyrus FHT in

the presence of 2-oxoglutarate and Fe2+ led to the formation of

the dihydroflavonols [14C]-DHK and [14C]-DHQ, respectively

(Fig. 1). No activity was observed when control enzyme

preparations from yeast containing the empty expression vector

Table 1

Effects of potential inhibitorsa of 2-ODDs on FHT and FLS activities

Code Compound

CH1 Calcium 3-hydroxy-5-oxo-4-propionyl-cyclohex-

3-ene-1-carboxylate (prohexadione-Ca)

CH2 3-Hydroxy-5-oxo-4-cyclopropanecarbonyl-cyclohex-

3-ene-1-carboxylic acid ethyl ester (trinexapac-ethyl)

CH3 3-Hydroxy-5-oxo-4-propionyl-cyclohex-

3-ene-1-carbothioic acid S-ethyl ester

CH4 3-Hydroxy-5-oxo-4-propionyl-cylohex-

3-ene-1-carbaldehyde

CH5 3-Hydroxy-5-oxo-4-butyryl-cyclohex-

3-ene-1-carboxylic acid ethyl ester

CH6 3-Hydroxy-5-oxo-4-propionyl-clohex-

3-ene-1-(2-dimethylamino)-thiazole

CH7 3-Hydroxy-5-oxo-4-propionyl-cyclohex-

3-ene-1-pentanoic acid

CH8 Sodium 4,6-Dioxo-2,2-dimethyl-5-

(1-alloxyamino-butylidene)-cyclohexane-1-

carboxylic acid methyl ester (alloxydim-Na)

CH9 3-Hydroxy-5-oxo-4-(1-ethoxyimino-butyl)-cyclohex-

3-ene-1-(tetrahydro-thiopyran-3-yl) (cycloxydim)

ACO Benzene-1,2,4,5-tetracarboxylic acid

Daz Succinic acid 2,2-dimethyl hydrazide (daminozide)

NCO1 Pyridine-2,4-dicarboxylic acid

NCO2 Pyridine-2,5-dicarboxylic acid

NCO3 Pyridine-2,6-dicarboxylic acid

NCO4 Pyridine-3,4-dicarboxylic acid

NCO5 Pyridine-3,5-dicarboxylic acid

NCO6 Pyridine-2,6-dicarboxylic acid chloride

NCO7 Pyridine-2,3-dicarboxylic acid (quinolinic acid)

NCO8 Pyrrole-3,4-dicarboxylic acid diethyl ester

NCO9 Pyrazole-3,5-dicarboxylic acid (hydrate)

NCO10 Piperidine-2-carboxylic acid

NCO11 Piperazine-1,4-bis-(2-ethane sulphonic acid)

NCO12 Piperidine-4-carboxylic acid

Enzyme activities were measured in the presence of potential inhibitors using recom

Enzyme activities are expressed as % rel. compared to a control (without inhibitor).

protein for FLS and 2.6 mkat kg�1 for DFR. The latter served as a control for unspeci

(heteroanalogous) carboxyl functions; ACO, aromate with carbonyl functions; Daza In order to improve clarity, the nomenclature used for CH1–CH9 has been made

always comply with IUPAC or CAS rules.

pYES were used. The Malus FHT reaction was optimized for

the screening assay. An optimum at pH 7.25 was observed for

naringenin and eriodictyol as substrates. Highest reaction rates

were measured at 30 8C, where the formation of DHK was

linear with time up to 15 min and with protein concentration up

to 17 mg protein in the assay. The values for apparent Km and

Vmax were 10 mM and 117.5 mkat kg�1 protein for naringenin

(Vmax/Km = 11.8 mkat kg�1 mM�1) and 12 mM and 102.9 mkat

kg�1 protein for eriodictyol (Vmax/Km = 8.6 mkat kg�1 mM�1).

Hence, a slightly higher preference was observed for naringenin

compared to eriodictyol.

3.2.2. Flavonol synthase (FLS)

The identity of the putative FLS-cDNA clone isolated from

Malus cv. ‘M9’ was also confirmed by assays with the

recombinant enzyme. Incubation of the enzyme preparations

with [14C]-DHK or [14C]-DHQ in the presence of 2-

oxoglutarate and Fe2+ led to the expected formation of the

Concentration

(mM)

FHT

(% rel.)

FLS

(% rel.)

DFR

(% rel.)

1/0.1 10/33 11/29 104/105

1/0.1 9/38 50/77 105/99

1/0.1 15/40 33/90 103/105

1/0.1 33/75 56/82 86/101

1/0.1 36/68 48/88 99/103

1/0.1 25/56 44/73 81/103

1/0.1 13/75 23/64 97/108

1/0.1 89/115 78/84 105/121

1/0.1 100/100 104/100 118/87

1/0.1 100/97 95/98 110/113

1/0.1 108/108 97/104 98/110

1/0.1 7/21 3/13 114/108

1/0.1 320/132 3/11 127/116

1/0.1 220/186 150/144 115/100

1/0.1 98/101 86/85 127/116

1/0.1 99/99 128/102 100/101

1/0.1 196/194 120/105 104/102

1/0.1 205/120 171/115 115/101

1/0.1 113/100 113/103 95/100

1/0.1 97/100 22/79 103/97

1/0.1 75/84 91/94 112/113

1/0.1 96/101 115/102 95/99

1/0.1 106/107 103/102 97/99

binant enzymes. Inhibitor concentrations are final concentrations in the assay.

Hundred percent correspond to 3.3 mkat kg�1 protein for FHT, 0.13 mkat kg�1 -

fic effects. CH1–CH9, cyclohexanediones; NCO1–NCO12, N-heterocycles with

, daminozide.

uniform with regard to numbering of the carbon atoms and, therefore, does not

H. Halbwirth et al. / Plant Science 171 (2006) 194–205 201

Table 2

Influence of the inhibitor/2-oxoglutarate ratio on the inhibitory effect of

selected compounds

Ratio inhibitor/

2-oxoglutarate

Relative FHT

activity (%)

Relative FLS

activity (%)

Controls – 100 100

NCO1 5:1 11 7

1:1 12 8

1:2 25 18

1:5 76 68

CH3 5:1 16 38

1:1 35 62

1:2 72 86

1:5 98 100

CH7 5:1 14 26

1:1 38 40

1:2 72 75

1:5 95 95

Enzyme activities were measured at a fixed concentration of 0.5 mM inhibitor

and 2-oxoglutarate concentrations from 0.1 to 2.5 mM. Relative activities were

calculated against controls without inhibitor.

flavonols kaempferol and quercetin, respectively (Fig. 1).

Formation of these products was not observed with enzyme

preparations from control yeast carrying the empty expression

vector. The enzyme also required 0.6 M sorbitol in the assay

buffer for measurable enzyme activity. An optimum at pH 7.25

was observed for DHK and DHQ as substrates. Highest reaction

rates were achieved at 30 8C, where the reaction was linear with

time for up to 30 min and 210 mg protein in the assay. The values

for apparent Km and Vmax were 1 mM and 18.5 mkat kg�1 protein

for DHK (Vmax/Km = 18.5 mkat kg�1 mM�1) and 1.2 mM

and 21.5 mkat kg�1 protein for DHQ (Vmax/Km = 17.9 mkat

kg�1 mM�1).

3.3. Screening for dioxygenase inhibitors

Selection of candidate compounds was based on structural

similarity to 2-oxoglutarate and also included some published

dioxygenase inhibitors (NCO1, NCO2, NCO3) [20,21]. Two

series of substances were screened: (i) cyclohexanediones such

as prohexadione-Ca (Fig. 2), mostly acyl-substituted and (ii)

pyridine dicarboxylic acids and other N-heterocycles with

carboxyl functions (Fig. 4). The common feature of most

selected compounds is the presence of two or three carbonyl

groups or heteroanalogous carbonyl functions. Some of the

compounds used for screening are components of commer-

cially available plant growth regulators (prohexadione-Ca,

CH1 and trinexapac-ethyl, CH2) [1], which served as positive

controls for inhibition, whereas CH8 and CH9 are gramini-

cides inhibiting acetyl-CoA-carboxylase in fatty acid bio-

synthesis [21].

Results obtained with the heterologously expressed FHT

from Pyrus turned out to be identical to those of heterologously

expressed FHT from Malus and, therefore, are not shown

separately. Possible influences of pH and buffer components

could be excluded since FHT and FLS assays were performed

with the same buffer system. Dihydroflavonol 4-reductase

(DFR, EC number 1.1.1.219) assays performed under the same

conditions were also included (Table 1). No significant

influence on this NADPH-dependent oxidoreductase, which

represents a different enzyme class, was observed, so that the

possibility of unspecific effects on enzyme activities such as

enzyme denaturation can be ruled out. As a result of this

screening, several compounds with distinct effects on the

dioxygenases FHT and FLS were identified (Table 1).

Unexpectedly, some compounds had a stimulating effect on

enzymatic activity in the in vitro assay.

3.4. Cyclohexanediones and related structures

Of the cyclohexanediones tested, CH1–CH7 inhibited FHT

and FLS activities at a concentration as low as 0.1 mM.

However, the extent of inhibition, was variable and depended

on compound structures. For CH2, CH3 and CH5, it can be

expected that the effects are different in vitro and in planta due

to the action of plant esterases, which can hydrolyze the ester

function [1]. The inhibition effect was overcome by increasing

the 2-oxoglutarate concentrations, which demonstrated the

competitive nature of the FHT and FLS inhibition (Table 2). For

prohexadione-Ca the IC50 was 23 mM for FHT and 50 mM for

FLS (Fig. 6).

For anthocyanidin synthase (ANS), which is closely related

to FLS and FHT [22], the binding mode of the co-substrate

2-oxoglutarate was revealed by a crystallographic study of an

enzyme/Fe2+/2-oxoglutarate/substrate analog complex [23]. It

could be shown that Fe2+ in the active centre is complexed by

the C1-carboxylate and the oxo group of 2-oxoglutarate, an

aspartyl, two histidinyl residues and a H2O ligand. In addition,

the negative charge of the C5-carboxylate of 2-oxoglutarate

interacts with the positive charge of an arginine residue. In the

case of the acylcyclohexanedione inhibitors (CH1–CH7), one

of the respective oxo functions of the ring corresponds to the

oxo group of 2-oxoglutarate. Similarly, the carbonyl oxygen of

the acyl residues in positions 1 and 4 substitute for the

carboxylate groups in positions 1 and 5 of 2-oxoglutarate

(Fig. 7A).

The general structural requirements for effective inhibition

are demonstrated in Fig. 7A. All compounds possessing a

3-hydroxy-5-oxo-cyclohex-3-ene (or the tautomeric 3,5-dioxo-

cyclohexane) structure with a carbonyl substituent in position 1

(R side: e.g. carboxylic or carbothioic acid or ester, CH1, CH2,

CH3, CH5; carbaldehyde, CH4; or electronegative thiazole,

CH6) and a second substituent in position 4 (R0 side: carbonyl

function provided by propionyl or related substituents) are

strong inhibitors. These findings correspond with reports on

inhibitors of 2-ODDs involved in gibberellin biosynthesis

[1,19,20]. Carbonyls of the acylcyclohexanediones may

interact via their negative partial charges or the oxo functions

may form an enolate as a superior ligand. Obviously, the

heteroatoms of some of the active compounds can take on the

oxygen functions.

Apart from possessing a carbonyl (or a heteroanalogous

carbonyl) moiety, substitution at position 1 of the cyclohex-

H. Halbwirth et al. / Plant Science 171 (2006) 194–205202

Fig. 6. Concentration dependence of FHT and FLS inhibition by selected inhibitors of 2-ODDs. Recombinant FHT and FLS were incubated with increasing

concentrations of prohexadione-Ca, NCO1, NCO2 and NCO5, respectively. FHT (triangles, dashed lines) and FLS (squares, full lines) activities are expressed as % of

control (no added inhibitor). Hundred percent correspond to 3.1 mkat kg�1 protein for FHT and 0.15 mkat kg�1 protein for FLS.

anedione ring appears to be relatively flexible for inhibiting

FHT and FLS in vitro. Compounds with substituents clearly

different from CH1 and CH2, e.g. CH3, CH4, CH5, CH6, CH7

have still considerable activity. One may assume that generally

these molecules are stabilized by keto-enol tautomerism (three

oxygen functions at the R0 side) and would, therefore, give a

particularly good similarity to 2-oxoglutarate. In case of CH7

with the carbonyl at the end of the n-pentanoic acid substituent,

a ring conformation is possible that brings the carbonyl close to

the cyclohexanedione ring.

Fig. 7. Structural features of acylcyclohexanedione inhibitors. (A) Left: cyclohexane

For substituents R and R0 see text. Centre: their structure (blue) corresponds to the s

flavonoid structure (DHK). Center: structural overlapping of a typical acylcyclohexan

no 3-hydroxy function (cf. Fig. 1)]. Right: structure of prohexadione.

In contrast, the graminicidal compounds CH8 and CH9

generally showed a low or no inhibitory effect. This is in line

with their low structural similarity to 2-oxoglutarate and

prohexadione (Fig. 2). In particular, the structures lack a second

carbonyl or carbonyl-like substituent next to the 4-position of

the cyclohexanedione structure (equivalent to the 4-propionyl

moiety of prohexadione-Ca), which could mimic the C5-

carboxylate of 2-oxoglutarate. Another important structural

difference to 2-oxoglutarate is the presence of an N-oxime

function in the herbicidal molecules. Furthermore, neither the

diones with in vitro activity on FHT and FLS show common structural features.

tructure of 2-oxoglutarate (green). Right: structure of 2-oxoglutarate. (B) Left:

edione (prohexadione) and the flavonoid DHK [NAR is identical to DHK but has

H. Halbwirth et al. / Plant Science 171 (2006) 194–205 203

Fig. 8. Structural similarities of selected pyridine dicarboxylic acids to 2-oxoglutarate. The structure of 2-oxoglutarate is shown in green. Structures of NCO1 (left),

NCO2 (center), NCO 3 (right) are shown in light blue. The position of nitrogen is marked in dark blue.

Table 3

Effect of activating compounds on FHT and FLS activity in the absence or

presence of the cofactor 2-oxoglutarate

Addition of 0.5 mM

2-oxoglutarate

Addition

of 1 mM

Relative FHT

activity (%)

Relative FLS

activity (%)

No – 10 7

Yes – 100 100

No NCO2 25 n.d.

Yes NCO2 320 n.d.

No NCO3 18 10

Yes NCO3 220 145

No NCO6 17 9

Yes NCO6 198 128

No NCO7 21 12

Yes NCO7 209 171

2-Oxoglutarate and other low molecular weight compounds were removed by

gel chromatography from the enzyme preparation and enzyme activities were

determined in the absence or presence of 0.5 mM 2-oxoglutarate. All assays

contained 0.1 mM Fe2+ and 0.4% potassium-ascorbate in the buffer. Relative

activities were determined against a control without inhibitor. Hundred percent

correspond to 3.3 mkat kg�1 protein for FHT and 0.13 mkat kg�1 protein for

FLS. n.d., not determined.

benzene derivative ACO nor daminozide (Daz) affected FHT

or FLS activity (Table 1), although Daz inhibits other 2-

oxoglutarate-dependent dioxygenases involved in gibberellin

biosynthesis [1].

Structural analyses demonstrated that particularly the 1-

acyl-substituted cyclohexanediones show pronounced struc-

tural similarity not only to the co-substrate, 2-oxoglutarate, but

also to the flavonoid substrates DHK and NAR (Fig. 7B). These

compounds can obviously interact with flavonoid dioxygenases

in two different ways. They can affect both the substrate and the

co-substrate sites and this may explain their relatively high

degree of inhibition. Absence (NAR) or presence (DHK) of the

3-hydroxyl group in the flavonoid substrate could account for

differing effects of some inhibitors on both enzymes (CH2,

CH3, and CH6). This 3-hydroxyl group overlaps with part of

the acyl residue of the acylcylohexadiones in structural

comparison.

3.5. Pyridine dicarboxylic acids and other N-heterocycles

with carbonyl functions

In previous investigations, certain pyridine dicarboxylic

acids (NCO1, NCO2) have been reported to inhibit other 2-

ODDs, e.g. two gibberellin 2b-hydroxylases [19] and prolyl 4-

hydroxylase involved in collagen biosynthesis [20]. In our

study, NCO1 strongly inhibited FHT and FLS (Table 1, Fig. 6;

IC50 25 mM for FHT, 2.3 mM for FLS). In contrast, strong

activation was observed unexpectedly for NCO2, NCO3,

NCO6 and NCO7, which increased FHT activity up to three

times (Table 1). Moreover, significant differences were

observed in some cases for FHT and FLS. In particular

NCO2 and NCO9 acted as strong inhibitors of FLS (NCO2:

IC50 80 mM, NCO9: IC50 70 mM), but either enhanced (NCO2)

or did not affect (NCO9) FHT activity (Fig. 6, Table 1). The

observed activating effects are in obvious contrast to a

competitive inhibition that was expected because of the

structural similarities of NCO1, NCO2 and NCO3 to 2-

oxoglutarate. The nitrogen atom of the respective pyridine

overlaps with different oxygen functions of 2-oxoglutarate

(Fig. 8). In contrast, no oxygen function of 2-oxoglutarate

corresponds to the nitrogen atom of NCO3 (Fig. 8).

Compounds that enhanced FHT and FLS activities were

also tested in case they could act as additional co-substrates.

Enzyme assays, in which 2-oxoglutarate was removed from the

protein extract by gel chromatography, showed strongly

reduced FHT or FLS activity (Table 3). However, addition

of the respective activating compounds did not result in

restored or even enhanced enzyme activity as found in the

presence of 2-oxoglutarate. It is remarkable, that assays

performed without addition of 2-oxoglutarate still showed a

remaining activity of approximately 10% (Table 3). We

assume that this is due to enzyme bound 2-oxoglutarate, which

co-purified with the 2-oxoglutarate-dependent enzymes. Such

an effect had been observed for FHT before (Forkmann,

unpublished).

On the other hand it could be shown for NCO3 that lower

Fe2+ concentrations led to higher activation (Table 4). Several

interactions are possible between enzyme, enzyme-bound

Fe2+, Fe2+ in solution, flavonoid substrate, 2-oxoglutarate and

the carboxylated N-heterocycles. Some dicarboxypyridines

showed structural similarities to the flavonoid substrates

H. Halbwirth et al. / Plant Science 171 (2006) 194–205204

Fig. 9. Structural similarities of selected flavonoids to various pyridine dicarboxylic acids. Structures of NAR (above) and DHK (below) (both shown in grey) are

compared with structures of NCO1 (left), NCO2 (center), NCO3 (right) (shown in light blue). The positions of nitrogen and oxygen in the rings are marked in dark

blue and in red, respectively.

DHK and NAR (Fig. 9). However, it can be assumed that

competition with the flavonoid substrate would lead to

inhibition rather than activation.

It is likely that the carboxylated N-heterocycles chelate Fe2+

in analogue to 8-hydroxyquinoline, which forms stable

complexes with many transition metal ions [24]. This could

explain the inhibitory action of these compounds. In the

crystallographic study of ANS, a decrease of the electron

density of Fe2+ was observed with citrate as a chelator in the

crystallization solution [22,23]. However, chelation would

lower the available concentration of Fe2+ as enzyme cofactor

and hence would be expected to reduce enzyme activity.

Flavonoids can also chelate Fe2+ [25–27] and excessive Fe2+

might decrease the availability of flavonoid substrates.

Addition of the carboxylated N-heterocycles as chelators

would liberate the weaker flavonoid chelators from the

flavonoid/Fe2+ complex and augment the availability of

flavonoids as enzyme substrates. As expected, the activating

effect is much stronger for low Fe2+ concentrations (Table 4).

Moreover, NAR as the weaker chelator (no vicinal O functions)

in comparison to DHK (vicinal 3-hydroxy/4-oxo functions) is

obviously released more easily by the competing chelators and

hence FHT activity is increased more strongly than FLS activity

(Table 4). In addition, FHT had a much higher activity in this

Table 4

Influence of the Fe2+ concentration on the activating effect of NCO3

Fe2+concentrations

(mM)

Relative FHT

activity (%)

Relative FLS

activity (%)

1000 186 144

100 220 169

10 250 187

1 300 202

Relative enzyme activities were measured at a fixed concentration of 0.1 mM

NCO3 and Fe2+ concentrations from 1000 to 1 mM. Relative activities refer to

control assays in the absence of inhibitor.

assay than FLS. Hence, better substrate availability led to a

stronger effect in case of the FHT assays. It can be concluded

that competitive Fe2+ chelation by flavonoid substrates and

activating compounds provides a plausible explanation for the

observed in vitro activation effects.

4. Conclusions

The screening revealed several potent dioxygenase

inhibitors, which were predominantly acylcyclohexane-

diones, and demonstrated their effects on the flavonoid

enzymes FHT and FLS. The primary structural features of

these compounds necessary for competitive interaction have

been identified in vitro. Structural comparisons of acylcy-

clohexadiones with flavonoid substrates showed that for these

dioxygenases competitive inhibition can also be expected at

the substrate binding site, beyond the general effect on the

2-oxoglutarate co-substrate binding site common to all such

dioxygenases. The inhibiting compounds identified may now

be tested for their in vivo effects to explore their potential in

agronomic applications.

N-Heterocyclic compounds were tested with respect to

effects on FHT and FLS as a second group of structural

analogues of 2-oxoglutarate. Several of these compounds are

also potent inhibitors, but in some cases the inhibitory effect

seems to be counteracted by their action as Fe2+ chelators,

which resulted in higher free flavonoid substrate concentrations

in vitro and, hence, to a higher substrate turn-over.

In contrast to prohexadione-Ca, which shows comparable

inhibitory effects on FHT and FLS, some of the compounds

tested show a higher inhibition of FHT than FLS, e.g. CH2,

CH3. These inhibitors could be of practical interest, because

the beneficial changes in the flavonoid spectrum would be

achieved with lower unintended effects on dioxygenases

other than FHT. Other FHT enzymes (e.g. from leaves of

grape vine, strawberry, rose) show a similar inhibition by

H. Halbwirth et al. / Plant Science 171 (2006) 194–205 205

prohexadione-Ca [3,4,7], and almost identical results were

obtained in this study with recombinant FHT from apple and

pear, which show some variation in amino acid sequences.

Given the known effects of prohexadione-Ca on 2-ODDs of the

gibberellin pathway, it appears likely that the competitive and,

hence, inhibitory effects of the new compounds on FHT and

FLS activity are transferable to other 2-oxoglutarate-depen-

dent plant dioxygenases, irrespective of a putative additional

competition with the flavonoid substrates in case of flavonoid

enzymes. Identification of a 2-ODD gene function in

benzoxazinoid biosynthesis in wheat and maize is a recent

example of application of an inhibitor to other 2-ODDs [28].

Therefore, the new inhibitors can also serve as further tools

to study enzyme activities of the wide-spread family of

2-ODDs.

Acknowledgements

Part of this work was supported by the European Commission

(QLK5-CT-1999-01583). K. Stich and H. Halbwirth gratefully

acknowledge the support by Vienna University of Technology

(GZ 9006.10/006/2005). C. Statnik and R. Paltram are

acknowledged for excellent technical assistance and B. Ros

for performing work on molecular cloning. Special thanks go to

Jurgen Greiner for his extensive assistance during the inhibitor

studies. Finally, we would like to thank U. Hesse for support in

structure comparisons, W. Heller for helpful discussion and E.

Meggeneder for critically reading the manuscript.

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