1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies
1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO3−) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO3−, and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO3− is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO3− and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO3− likely plays a major role in the stabilization of the substrate in the active pocket.
KeywordsEnzyme kinetics Fluorescence Docking 1-Aminocyclopropane-1-carboxylic acid oxidase Nonheme iron
The X-ray structure of ACCO from Petunia hybrida has been solved, and revealed a core composed of β-strands folded into a distorted jelly-roll motif named double-strand β-helix fold or cupin fold . The Fe(II) is coordinated to the side chains of H177, H234, and D179 in a two-histidine, one-aspartate facial triad. However, no crystallographic data on the binding of the different substrates/cofactors at the active site are yet available. Moreover, several pieces of evidence obtained from mutagenesis studies have suggested that this structure might not be an active form of the enzyme [8, 9, 10]. With use of these findings, a structural model of apple ACCO (Malus domestica) displaying a different conformation has been constructed [9, 11].
On the basis of above-mentioned structural motifs (double-strand β-helix fold, facial triad) ACCO is related to the 2-oxoglutarate (2-OG)-dependent dioxygenases, a large family of Fe(II)-containing enzymes which for the most part utilize 2-OG as a cosubstrate to catalyze a wide range of oxidation reactions [12, 13, 14].
It is generally accepted that the conversion of ACC into ethylene proceeds via a radical mechanism [15, 16, 17]. EPR/electron–nuclear double resonance studies have suggested that the first step of the reaction consists of the coordination of ACC to the iron in a bidentate manner via the nitrogen of the amine and an oxygen of the carboxylate group [18, 19]. There are only few spectroscopic data on the interaction mode of the other cofactors/substrates at the active site, and several catalytic mechanisms have been proposed that all involve an Fe(IV)=O but differ regarding the nature of the substrate oxidizing species [20, 21].
In the 1980s investigators observed that CO2 promotes ACC-dependent ethylene production in plant tissues . Dong et al.  reported that CO2 also activates ACCO in vitro. The role of this cofactor, its interaction at the active site, and its mode of action are still unknown. It has been shown that HCO3− prevents the catalytic inactivation of the enzyme, and more specifically that it has a protective role against ACC-dependent inactivation processes [24, 25, 26]. Alternatively, bicarbonate has been proposed to facilitate a proton transfer step during dioxygen activation . It is not fully clear whether CO2 or HCO3− is the activator/protector. On the basis of inhibition studies, Rocklin et al. , however, concluded that HCO3− could be the active form at the catalytic center.
Materials and methods
All chemicals were purchased from Sigma-Aldrich except for ACC and AMEP, which were purchased from Acros Organics. The synthesis of ACP was performed following our previously described procedure .
Overexpression and purification of recombinant tomato ACCO
Plasmid PET21a (Novagen) containing the ACCO gene (pTOM13) from Lycopersicum esculentum was donated by the group of C. Schofield from Oxford University (UK). ACCO was produced in Escherichia coli strain BL21(DE3) and purified according to already described procedures [8, 33, 34]. Cells (2 L) were grown in flasks at 37 °C (200 rpm) in terrific broth containing 100 μg/mL ampicillin to an optical density at 600 nm of approximately 1. The temperature was then adjusted to 28 °C and the culture was grown for 40 min, after which protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (0.5 mM) and cells were harvested after 3 h. Purification steps were performed at 4 °C. Thawed cell pellets were resuspended in buffer containing 25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) at pH 8.0, 10 % glycerol, 3 mM EDTA, 5 mM dithiothreitol, 1 mM benzamidine 10 μM leupeptine, 1 μg/mL pepstatine, and 0.8 mM Pefabloc® SC (Roche Applied Science). Lysis was performed using a French® press (Thermo Scientific). Cell debris was eliminated by centrifugation at 20,000 rpm for 1 h (Sorvall SS34). The supernatant was loaded on a (diethylamino)ethyl (DEAE) Sepharose resin (DEAE FF, GE Healthcare) equilibrated with buffer A (25 mM HEPES pH 8.0, 10 % glycerol, 3 mM EDTA, 1 mM benzamidine) and was eluted with buffer A containing 100 mM NaCl. ACCO was 90–95 % pure according to sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis.
Activity assays with ACCO
Assays to measure ethylene production from ACP were performed using 18 mM NaHCO3, 8 mM l-ascorbic acid, and 40 μM Fe(SO4)2(NH4)2·6H2O with various quantities of ACCO ranging from 5 to 100 μg. Concentrations of ACP ranging from 0.06 to 2.4 mM were used, and ethylene was measured after reaction times ranging from 10 to 60 min. Ethylene production from ACP in the absence of enzyme was evaluated under the same conditions.
Measurements were performed with a Shimadzu GC-2014A gas chromatograph equipped with a Porapak Q 80/100 column (0.125 in.) and a flame ionization detector. The following conditions were used: N2 carrier gas, Tinjector = 250 °C, Toven = 100 °C, Tdetector = 250 °C. Ethylene was quantified versus an external standard (1 % ethylene in nitrogen, Alltech).
Fluorescence measurements were performed at 20 °C using a Horiba Jobin–Yvon Fluoromax-4 spectrometer. The excitation wavelength was 290 nm (1-nm slit widths), and the emission wavelength was 345 nm (5-nm slit widths). Titration experiments were performed using 0.65 μM enzyme in 25 mM HEPES buffer (pH 7.2) that had been prepared anaerobically in a glove box. The quartz cuvettes used were sealed with a septum and cofactors were added using a gastight syringe in portions of 1–20 μL from concentrated stock solutions stored under an inert atmosphere. Titration experiments were performed more than three times and up to seven times for each condition. When necessary, the cofactors were used at the following concentrations: 30 μM Fe(II), 18 mM bicarbonate, and 1 mM ACC.
The structural model of tomato ACCO was obtained using the modeled structure of ACCO from M. domestica as a template . The sequences alignment between the ACCO from M. domestica and the one from tomato was generated using ClustalW . The two enzymes display 77.8 % sequence identity on 315 amino acids. Homology modeling was performed with Modeller 4.0  and the geometry around the iron was constrained with respect to the values observed in the X-ray structure of petunia ACCO (Protein Data Bank code 1WA6) .
Docking of the designed compound was performed using the AutoDock Tools 4.2 program package [39, 40]. Ligand coordinates and charges (modified neglect of differential overlap calculations) were generated with HyperChem 8.0. Ligand active torsions were defined by the AutoTors tool of the AutoDock package. Kollman charges were applied and nonpolar hydrogen atoms from the macromolecule were merged to their supporting atoms. To compensate for the overestimation of the electrostatic contribution of the iron ion, its charge was set at +0.8 in accordance with Hu and Shelver  and Langella et al. . A 60 × 60 × 60 points affinity grid was centered on iron with a spacing of 0.375 Å, and 255 runs were performed. Different random seeds were used for each of the 255 runs. The resulting docked conformations of the ligand were clustered according to the default AutoDock scoring function using a root mean square deviation (RMSD) of 2 Å. In the case of ascorbate, the RMSD was set at 3 Å.
The ability of ACCO to oxidize various cyclic analogs of ACC to ethylene and various acyclic amino acid substrates to the corresponding carbonyl compound, CO2, and ammonia has been reported in the literature [28, 29, 31, 43, 44]. This ability has also been observed recently using a functional model complex of ACCO . The α-aminophosphonate derivatives ACP and AMEP constitute a pair of new substrate analogs/inhibitors of ACCO. Therefore, we first investigated the oxidation of these molecules. With use of buffered solutions of ACP containing Fe(II), ascorbate, and bicarbonate, small quantities of ethylene were produced. This ethylene production was found (1) to be linear with ACP concentration, reaching a maximum of 0.2–0.3 nmol ethylene after 20 min of reaction when starting from 200 nmol ACP (i.e., 0.1–0.15 % conversion yield), (2) to be independent of the presence of ACCO, i.e., no additional oxidation of ACP was detected in the presence of up to 100 μg ACCO, and (3) to be independent of the presence of bicarbonate. No measurable oxidation of AMEP to acetone was detected.
Apparent kinetic constants for 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) and inhibition types and constants for the inhibition of the activity by 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP)
11.3 ± 0.5
10.8 ± 0.2
11.9 ± 0.4
0.15 ± 0.02
3.3 ± 0.4
2.7 ± 0.4
1.5 ± 0.1
0.5 ± 0.1
1.6 ± 0.4
2.3 ± 0.4
1.6 ± 0.2
3.6 ± 0.4
Determination of binding affinities by fluorescence quenching measurements
Metal and 1-aminocyclopropane-1-carboxylic acid (ACC) dissociation constants determined by intrinsic fluorescence quenching using several conditions
Kd of Fe(II) (μM)
Kd of ACC (μM)
3.1 ± 0.3
Apo-ACCO + 1 mM ACC
3.0 ± 0.5
Apo-ACCO + 18 mM HCO3−
2.9 ± 0.5
Apo-ACCO + 1 mM ACC + 18 mM HCO3−
1.2 ± 0.1
Apo-ACCO + 30 μM Fe
253 ± 9
Apo-ACCO + 30 μM Fe + 18 mM HCO3−
98 ± 4
When ACC was added to apo-ACCO, no clear fluorescence change was observed. In the presence of 30 μM Fe(II), ACC led to approximately 30 % additional quenching of the fluorescence with, again, no significant changes in the emission maximum. Fluorescence quenching upon addition of ACC was followed (Fig. 2) and simulations were performed to obtain the dissociation constants (Table 2). The dissociation constant of ACC in the absence of bicarbonate is 253 μM. The binding of ACC is improved about 2.5-fold in the presence of 18 mM bicarbonate, and the dissociation constant determined under these conditions is 98 μM. The dissociation constant is in rather good correlation with the value of 150 μM for KM determined here for this enzyme (Table 1) as well as for the value of 77 μM for KM reported in the literature .
When bicarbonate was added to apo-ACCO in the presence or absence of other substrates/cofactors, including Fe(II), no significant fluorescence changes were observed and no dissociation constant for this cofactor could be obtained using this technique. Finally, the binding of ascorbate was not examined by this approach because this substrate absorbs at the excitation wavelength, thus interfering with the analysis.
Modeled structure for tomato ACCO
Docking experiments: substrates
Docking HCO3− to an ACCO/Fe enzyme led to a single cluster of solutions as well, in which the bicarbonate ion is found coordinated to the Fe(II) at the place of the carboxylate group of ACC. To avoid collapsing of this cofactor on the iron during the calculations, docking of HCO3− was performed on the ACCO/Fe/ACC complex obtained from the previously described calculations. Again, a single cluster of solutions was obtained (Fig. 3). HCO3− was found at distances allowing interactions (i.e, hydrogen bonds or electrostatic interactions) with several residues in the active site: K158 and R300 on one side, and S246, R244, and Y162 on the other side. In addition, a hydrogen bond between the OH group of HCO3− and the noncoordinated oyxygen atom of ACC was found.
Although at pH 7 ascorbate exists mainly as a deprotonated form in solution, we investigated the binding of both the ascorbate and ascorbic acid forms to ACCO. Docking to the ACCO/Fe complex and on ACCO/Fe/ACC and ACCO/Fe/ACC/HCO3− models was performed. Many clusters of conformations were found and we thus increased the RMSD to 3 Å for the clustering. After elimination of improbable solutions, in particular when this substrate was found coordinated to the metal ion, in contradiction with spectroscopic data , we ended up with one possible conformation, common to all our calculations and independent of the protonation state. In this solution, the most remarkable interactions are found with R300 opposite to HCO3− and with the amide groups of the peptide bonds of R175, R235, and V236.
Docking experiments: inhibitors
α-Aminophosphonates display acidity constants for the protons of the phosphonic acid moiety in the ranges pK1 = 2.3–2.5 and pK2 = 5.5–7 . Taking into account that the kinetic data were obtained at pH 7.2, we performed docking of ACP and AMEP in two different protonation states of the noncoordinated oxygen atom of the phosphonate group (Scheme 2). Docking either the monodeprotonated form or the fully deprotonated form of ACP to ACCO/Fe led to a similar position (Fig. 4). In this model, ACP is coordinated in a bidentate mode to the metal ion, slightly twisted as compared with the position of ACC. The amine and the phosphonate groups are located roughly at the positions of the amine and carboxylate groups of ACC, respectively. In addition, one oxygen atom of the phosphonate group is found at a distance allowing interaction with K158 and R300.
To evaluate the influence of ACP on the binding of bicarbonate, docking of this cofactor was performed on the ACCO/Fe/ACP complex obtained from the above-mentioned calculations, with the fully deprotonated phosphonate. This led to a single cluster of solutions where HCO3− is found at hydrogen-bond distance from a noncoordinated oxygen atom from the phosphonate group and in a position slightly different from that obtained with ACC. The distances to K158 and R300 are longer, consistent with the interaction of these residues with ACP rather than with bicarbonate. It is worth mentioning that from our calculation, the position of ascorbate was not influenced by the substitution of ACC with the phosphonate analog.
Docking experiments with the fully deprotonated AMEP led to a conformation in which the inhibitor is bound in a bidentate manner on the iron ion, similar to that obtained with ACP or ACC. Docking of the protonated form of AMEP led to many clusters of solutions (14 conformations). Apart from the case where the inhibitor is found coordinated to the metal ion, there are significant numbers of solutions where the inhibitor is interlocking with the binding site of ascorbate.
The enzyme ACCO is a complex system requiring the presence of many substrates/cofactors to perform the synthesis of ethylene from ACC. In addition, the enzyme is largely inactivated during catalysis, making mechanistic studies particularly difficult [24, 25]. These peculiarities require, may be more so than with other enzymes, the use of a combination of techniques to investigate structure–function relationships. Accurate structural data are also of particular interest, and so far only one crystal structure of ACCO has been reported from P. hybrida (Protein Data Bank code 1WA6), with no data on the binding of the different cofactors at the active site . Although active as a monomer, ACCO appears as a homotetramer in the crystal form with the C-terminal part directed away from the active site and interlocking with the C-terminal part of an adjacent monomer. Several pieces of evidence obtained from mutagenesis studies have, however, suggested that this C-terminus may play a role in catalysis [8, 9]. In addition, although important for activity , the R244 residue belonging to an RXS motif exclusively conserved in the structural subfamily of ACCO  is directed away from the metal in the crystal form. Taking into account the possible irrelevance of the crystallographic structure of P. hybrida, we constructed a model structure for tomato ACCO based on the model developed by Yoo et al.  for apple ACCO. In this model, the C-terminus of ACCO is folded towards the protein core and the R244 residue points towards the metal.
The mechanism of fluorescence quenching upon metal binding is mainly described as a long-range energy transfer to an absorption band produced by the metal–protein interaction, therefore giving rise to significant loss of emission intensity [51, 52]. Alternative quenching mechanisms associated with metal binding have also been proposed . ACCO contains three tryptophan residues (W31, W86, and W203) located at distances ranging from 10 to 20 Å from the Fe(II) in the active site. Although the tryptophan residues are relatively far from the active site, the observed quenching of the tryptophan fluorescence upon iron binding (approximately 30 % decrease of intensity) is strong enough for the dissociation constants to be extracted. Quenching of fluorescence is also strong when ACC is added to ACCO in presence of iron. The fact that no significant fluorescence quenching is observed when bicarbonate is added to metalated ACCO is consistent with the fact that this cofactor probably does not interact with the metal ion. On the other hand, it appears that bicarbonate influences the binding of ACC in the presence of iron by increasing the affinity of the substrate for the active site (Kd is 253 and 98 μM in the absence and the presence of bicarbonate, respectively), confirming the results previously reported by Zhou et al.  with avocado ACCO. In addition, the dissociation constant for the Fe(II) ion in apo-ACCO is 3.1 μM and is not modified by the presence of either ACC or HCO3−. It is only when ACC and bicarbonate are present together that the dissociation constant for the metal is decreased 2.5-fold to 1.2 μM. Our differential fluorescence results thus suggest that the stabilization of ACC in the active pocket by the bicarbonate cofactor increases the stability of the metal ion complex. Docking ACC on our tomato ACCO model suggested that ACC is coordinated to the metal ion in a bidentate fashion, via the nitrogen and one oxygen, in agreement with the results of spectroscopic studies [18, 19], as well as with the structures of several metal–ACC bioinspired complexes [54, 55, 56, 57]. Rocklin et al.  have suggested that the active form of the CO2 cofactor is HCO3−, and we used this form in our calculations. The binding site that we propose for HCO3− involves interactions with residues K158, Y162, R244, and S246 from the RXS motif and R300 from the C-terminus. The importance of the C-terminal residues for catalysis has already been stressed, in particular through the potential involvement of R300 in HCO3− binding . Our calculations also highlight a possible hydrogen-bonding of HCO3− to the substrate ACC. This is consistent with the fact that ACC, which does not develop significant interactions with any amino acid in the active pocket, appears to be stabilized by the bicarbonate cofactor as observed in our experiments (see above). Any destabilization of the bicarbonate binding would therefore directly affect the efficiency of ACCO. Like us, Bassan et al.  have suggested that K158 could be involved in bicarbonate binding; yet mutations of K158 to alanine or cysteine almost completely abolish activity in kiwifruit ACCO . Similarly, it has been reported that mutations of residues R244 and S246 decrease the activity of the enzyme and increase KM for ACC . In addition to this stabilization role, the twofold to 290-fold decrease of Vmax observed for those latter mutants  suggests that bicarbonate also plays an important role during catalysis, possibly assisting a proton transfer in the dioxygen activation pathway . Our positioning of HCO3− is in complete agreement with its potential role in the promotion of the enzyme activity and its influence on the reaction rate [20, 23]. Mirica and Klinman  have suggested that a proton (or hydrogen atom) transfer occurs during the rate-limiting step of dioxygen activation by ACCO, but the source of the proton was not unambiguously determined. Anchored 5–7 Å away from the metal center by K158 among other residues, the bicarbonate cofactor would be perfectly located to assist a proton transfer in the catalytic mechanism. Finally, it is known that bicarbonate ions have a protective effect on inactivation processes of ACCO, as specifically observed in the presence of ACC . A simple mechanical effect of bicarbonate ion on the positioning of the substrate in the active pocket could therefore be crucial to avoid auto-oxidation reactions. Our interaction model is thus consistent with most of the observed effects for bicarbonate (stabilization, activation, and protection) and underlines that the role of this cofactor might be multifaceted and complex.
Even though additional pieces of experimental evidence would be required, a position where ascorbate is interacting with R300 and with the peptide bond of R175, R235, and V236 can be proposed. This position is different from the binding site involving interactions with R244 and S246 proposed by Yoo et al. . These authors docked the different cofactors of ACCO on an ACCO/Fe(II)–NO complex, in which the NO position on the iron was fixed trans to D179 by analogy with the isopenicillin N synthase (IPNS)/Fe–NO structure . In 2-OG oxygenases, the position trans to the aspartate is often occupied by the 2-oxo group of the 2-OG cofactor [12, 13, 14]. In our model, as mentioned above, the position trans to D179 is occupied by ACC. Consequently, the position proposed by Yoo et al. for ascorbate was never found among the most probable ones in terms of energy and/or clustering in our calculations.
Finally, it is worth noting that the results obtained from docking experiments performed on the crystal structure could not explain the experimental data and the role of the bicarbonate cofactor, and more particularly the observed stabilization effect on ACC binding, therefore strengthening our decision to use a modeled structure.2
Although structurally very similar, the α-aminophosphonates surprisingly displayed different behaviors. Whereas each of them is a competitive inhibitor of ACCO versus both ACC and HCO3−, ACP is a noncompetitive inhibitor and AMEP is a competitive inhibitor versus ascorbate. The latter finding is consistent with the finding of Brunhuber et al. , who reported that AIB, which shares its acyclic structure with AMEP, is a competitive inhibitor of ACCO versus ascorbate. Docking ACP on the metalated ACCO supports a coordination of ACP to the metal ion in a bidentate mode which is similar for the two possible protonation states of the inhibitor. There are only a few structural data on the bidentate coordination of α-aminophosphonate to metal ions . However, the bidentate binding of phosphonate-containing substrates observed in several nonheme iron-containing enzymes, such as 2-hydroxyphosphonic acid epoxidase [61, 62] and hydroxyethylphosphonate dioxygenase , strengthens our conclusion. From our calculation, one oxygen atom of the phosphonate group of ACP is interacting with K158 and R300. As a consequence, when HCO3− is docked on the ACCO/Fe/ACP complex, its position is modified as compared with that obtained with ACC, and in particular the interactions with K158 and R300 are weakened. Our results suggest that ACP interlocks with the binding of both ACC and HCO3−, providing a possible explanation for the results of the inhibition experiments in which ACP was found to be a competitive inhibitor versus both ACC and HCO3−. In this picture, the position of ascorbate is away from the binding site of ACP. This is also in agreement with our kinetic data, which suggest that its presence does not affect much the binding of ACP (pure noncompetitive inhibition pattern). The situation with AMEP appears slightly different, in particular with its protonated form. Indeed, unlike the deprotonated form that is found on the metal ion in the same position of ACP, the protonated AMEP can occupy different locations and in particular the site where ascorbate potentially binds. This could explain the different inhibition pattern observed between the cyclic and the acyclic α-aminophosphonate molecules, and in particular the fact that AMEP appears as a competitive inhibitor of ACCO versus ascorbate.
In conclusion, combining kinetic analysis of ACCO with two substrate analogs, fluorescence experiments, and theoretical studies, this work investigated the binding of the cofactors and more particularly of bicarbonate ions at the active site as well as a possible role for this cofactor. The proposed binding site for HCO3− involves interactions with several residues, such as that from the RXS conserved motif, K158, and a residue from the C-terminus (R300). In addition, our calculations evidence hydrogen bonding of this cofactor to the substrate ACC, in agreement with the observed substrate and metal stabilization in the active pocket from fluorescence quenching measurements. Interestingly, the position of the carboxylate function of ACC is similar to that of the 2-oxo function of 2-OG in related enzymes from the 2-OG-dependent dioxygenases [12, 13, 14]. This drives HCO3− (via hydrogen bonding) to a location corresponding to that of the terminal carboxylate of 2-OG. Finally, this substrate stabilization in the active pocket could also explain the protective role of HCO3− against inactivation processes, and the location of this cofactor is consistent with its participation in a proton transfer during catalysis.
For square-pyramidal geometry τ = 0 and for trigonal bipyramidal geometry τ = 1.
Docking experiments were also performed on the crystal structure (using a monomer from the tetramer and removing the phosphate molecule bound on the metal ion). The position of ACC is similar to that obtained on the modeled structure, although this is twisted. The amine group of ACC is found trans to H177 and the oxygen is coordinated approximately trans to D179. When HCO3− is docked on the ACCO/Fe/ACC complex from the above-mentioned calculations, the most favorable position is located far from ACC (more than 10 Å).
This work was supported by a grant from the Agence Nationale de la Recherche (ANR-09-JCJC-0080). The authors acknowledge C. Schofield and Z. Zhang from Oxford University for providing the plasmid containing the gene encoding tomato ACCO as well as for useful advice.
- 7.Stella L, Wouters S, Baldellon F (1996) Bull Soc Chim Fr 133:441–455Google Scholar
- 10.Dilley DR, Kadyrzhanova DK, Wang Z (2001) Acta Hortic 553:143–144Google Scholar
- 15.Adlington RM, Baldwin JE, Rawlings BJ (1983) J Chem Soc Chem Commun 290–292Google Scholar
- 16.Baldwin JE, Jackson DA, Adlington RM, Rawlings BJ (1985) J Chem Soc Chem Commun 206–207Google Scholar
- 17.Pirrung MC (1999) Acc Chem Res 32:711–718Google Scholar
- 45.Baráth G, Kaizer J, Sándor Pap J, Speier G, El Bakkali-Taheri N, Simaan AJ (2010) Chem Commun 46:7391–7393Google Scholar
- 48.Dunning Hotopp JC, Auchtung TA, Hogan DA, Hausinger RP (2003) J Inorg Biochem 93:66–70Google Scholar
- 49.Addison AW, Rao TN, Reedijk J, van Rijn J, Veschoor GC (1984) J Chem Soc Dalton Trans 1349–1356Google Scholar
- 54.Ghattas W, Gaudin C, Giorgi M, Rockenbauer A, Simaan AJ, Réglier M (2006) Chem Commun 1027–1029Google Scholar