JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 6, pp 939–949 | Cite as

1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies

  • Lydie Brisson
  • Nadia El Bakkali-Taheri
  • Michel Giorgi
  • Antoine Fadel
  • József Kaizer
  • Marius Réglier
  • Thierry Tron
  • El Hassan Ajandouz
  • A. Jalila Simaan
Original Paper

Abstract

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.

Keywords

Enzyme kinetics Fluorescence Docking 1-Aminocyclopropane-1-carboxylic acid oxidase Nonheme iron 

Introduction

The plant hormone ethylene is essential for many aspects of plant life, including root development, germination, senescence, fruit ripening, and defense mechanisms [1, 2]. In 1979, Adams and Yang [3] reported that ethylene is directly biosynthesized from 1-aminocyclopropane-1-carboxylic acid (ACC), a metabolite of methionine. This step is catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO), and the first complementary DNA encoding ACCO was obtained from tomato fruit by Hamilton et al. [4, 5]. The conversion of ACC into ethylene requires the presence of ferrous ions, dioxygen, and ascorbate (Scheme 1) [6, 7]. In addition, ACCO also requires the presence of CO2 (or bicarbonate, HCO3) for activity.
Scheme 1

Reaction catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). Asc ascorbate

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 [8]. 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 [22]. Dong et al. [23] 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 [27]. It is not fully clear whether CO2 or HCO3 is the activator/protector. On the basis of inhibition studies, Rocklin et al. [20], however, concluded that HCO3 could be the active form at the catalytic center.

The use of substrate analogs is a powerful strategy to obtain information on an enzymatic system, on the interaction mode of the substrates at the active site, and on the catalytic mechanism. Several groups have successfully employed this approach in the case of ACCO [28, 29, 30, 31]. α-Aminophosphonic acids constitute a family of new potential inhibitors of ACCO that can be used as surrogates for the corresponding α-aminocarboxylic acids. In this study, we used tomato ACCO and investigated the kinetic behavior of α-aminophosphonic acid analogs of ACC and α-aminoisobutyric acid (AIB), a commonly studied inhibitor of ACCO: 1-aminocyclopropane-1-phosphonic acid (ACP) [32] and (1-amino-1-methyl)ethylphosphonic acid (AMEP), represented in Scheme 2. The potential of these compounds as inhibitors was established. Binding constants and interactions between the cosubstrates/cofactors were investigated using tryptophan fluorescence quenching experiments. Finally, modeling and docking experiments were performed to rationalize the experimental data and to obtain a better insight into the interaction of the different cofactors and the inhibitors at the active site. On the basis of these experimental and theoretical approaches, a picture of the interactions at the active site is proposed as is a possible role for the bicarbonate cofactor.
Scheme 2

Chemical structures of the substrate and the inhibitors. a 1-aminocyclopropane-1-carboxylic acid (ACC), b 1-aminocyclopropane-1-phosphonic acid (ACP), and c (1-amino-1-methyl)ethylphosphonic acid (AMEP). Different protonation states of ACP and AMEP used in the docking experiments

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 [32].

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

Standard assays were performed at 29 °C in 1.7-mL hermetically sealed vials. The total assay volume was 200 μL and contained 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH 7.0 with 10 % glycerol, 5 μg ACCO, 18 mM NaHCO3, 8 mM l-ascorbic acid, 40 μM Fe(SO4)2(NH4)2·6H2O, and 1.25 mM ACC. Dioxygen was kept at 240 μM for all experiments (air-saturated water at 29 °C). After 7 min of reaction, 1 mL of the headspace gas was removed using a gastight syringe and ethylene production was quantified by gas chromatography. The rates were in the linear range (see the electronic supplementary material). Measurements were performed by varying the concentration of one substrate while maintaining the concentrations of the other substrates: 18 mM NaHCO3, 8 mM l-ascorbic acid, 40 μM Fe(II), and 1.25 mM ACC. The variation ranges were 0–1.5 mM for ACC, 0–8 mM for ascorbate, and 0–18 mM for NaHCO3. Nonlinear curve fitting to the Michaelis–Menten equation (Eq. 1) was performed using GraphPad Prism 4.0 to determine apparent kinetic constants:
$$ v = \frac{{V_{\max } [\hbox{S}]}}{{K_{\rm M} + [\hbox{S}]}},$$
(1)
where [S] is the substrate concentration.

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.

Inhibition studies

The inhibition studies were performed in 50 mM MOPS buffer containing 10 % glycerol at pH 7.0. Measurements were performed in the presence of an inhibitor, and the concentration of one substrate/cofactor was varied while maintaining the other substrates/cofactors at their optimal concentrations. The ACC concentration was varied from 0 to 1.5 mM, the ascorbate concentration was varied from 0 to 8 mM, the NaHCO3 concentration was varied from 0 to 18 mM, and iron was kept at 40 μM. Phosphonate derivatives were added at concentrations ranging from 0 to 4 mM. Each point was measured more than five times and the experiments were reproduced using an enzyme originating from a different purification. The inhibition patterns were determined using nonlinear curve fitting using Eqs. 2, 3, 4, and 5:
$$ v = \frac{{V_{\max } [\hbox{S}]}}{{K_{\rm M} \left( {1 + \frac{[I]}{{K_{\rm i} }}} \right) + [\hbox{S}]}}\quad{\text{for competitive inhibition}},$$
(2)
$$ v = \frac{{V_{\max } [\hbox{S}]}}{{K_{\rm M} + [S]\left( {1 + \frac{[I]}{{L_{\rm i} }}} \right)}}\quad{\text{ for uncompetitive inhibition}},$$
(3)
$$ v = \frac{{V_{\max } [\hbox{S}]}}{{K_{\rm M} \left( {1 + \frac{[I]}{{K_{\rm i} }}} \right) + [\hbox{S}]\left( {1 + \frac{[I]}{{L_{\rm i} }}} \right)}}{\text{ for mixed noncompetitive inhibition}},$$
(4)
$$ v = \frac{{V_{\max } [\hbox{S}]}}{{(K_{\rm M} + [\hbox{S}])\left( 1 +{\frac{ [I]}{{K_{\rm i} }}} \right)}}{\text{ for pure noncompetitive inhibition }}K_{\rm i} = L_{\rm i}, $$
(5)
where Ki and Li are defined as the dissociation constants for the enzyme–inhibitor and the enzyme–substrate–inhibitor complexes, respectively, [S] is the substrate concentration, and [I] is the inhibitor concentration. The set of curves for each substrate was fit simultaneously. The goodness of fits to each equation was used to assess the appropriate inhibition pattern. The inhibition constants and standard errors were derived from the fits. Inhibition types were confirmed by using Lineweaver–Burk representations as well as by using the method proposed by Cornish-Bowden [35].

Gas chromatography

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

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.

Fluorescence changes were analyzed by curve fitting (GraphPad Prism 4.0) according to Eq. 6 [36]:
$$ \Updelta I = I_{0} - I = \Updelta I_{\max } \frac{{\left( {K_{\text{d}} + E_{\text{T}} + L_{\text{T}} } \right) + \sqrt {\left( {K_{\text{d}} + E_{\text{T}} + L_{\text{T}} } \right)^{2} - 4E_{\text{T}} L_{\text{T}} } }}{{2E_{\text{T}}}},$$
(6)
where ET is the enzyme concentration, LT is the cofactor concentration, Kd is the dissociation constant, and ΔImax is the maximum fluorescence quenching at high cofactor concentration (infinite limit). Kd and ΔImax were obtained from curve-fitting analysis.

Structure modeling

The structural model of tomato ACCO was obtained using the modeled structure of ACCO from M. domestica as a template [8]. The sequences alignment between the ACCO from M. domestica and the one from tomato was generated using ClustalW [37]. The two enzymes display 77.8 % sequence identity on 315 amino acids. Homology modeling was performed with Modeller 4.0 [38] 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) [8].

Docking experiments

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 [41] and Langella et al. [42]. 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.

Results

Kinetic studies

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 [45]. 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.

Inhibition studies were performed with the phosphonate analogs ACP and AMEP. KM of tomato ACCO for dioxygen has been determined to be 28 μM [33]. In air-saturated water at 29 °C, the dioxygen concentration is approximately 240 μM. Although not fully saturating (only 90 % saturating), the effect of this cofactor was not investigated and the reported constants are considered as apparent. Initial velocities for the inhibition of ACCO by ACP with ACC, HCO3, or ascorbate as the substrate for which the concentration was varied are displayed in Fig. 1. Inhibition types were determined by fitting simultaneously the set of curves with the different equations (Eqs. 2, 3, 4, 5) and confirmed by using Lineweaver–Burk representations (see the electronic supplementary material). In the best solution, it appears that ACP is a competitive inhibitor versus both ACC and HCO3 but a noncompetitive inhibitor versus ascorbate (i.e., ACP binding to the enzyme seems hardly affected by the presence or the absence of ascorbate). The data obtained at high inhibitor concentration (2–4 mM) deviate from the model when ACC is the substrate for which the concentration was varied, suggesting a probably more complex ACCO–ACP interaction mode. The curve-fitting analysis was therefore performed without considering the highest concentration of ACP (4 mM) and without considering two points from the data set where the ACP concentration is 2 mM (filled in orange Fig. 1). The inhibition constants are reported in Table 1. The inhibition pattern obtained with AMEP is similar to that obtained with ACP when the concentrations of ACC and HCO3 were varied (see the electronic supplementary material). AMEP is a structural analog of AIB, which has been described as a competitive inhibitor of ACCO versus ACC with an inhibition constant of approximately 2 mM [28, 29, 30]. Here, the inhibition constants measured for the two phosphonate analogs against ACC are of the same order of magnitude (Ki of 1.5 and 2.3 mM for ACP and AMEP, respectively). However, ACP and AMEP can also interact at bicarbonate’s binding site with rather good affinity (Ki of 0.5 and 1.6 mM for ACP and AMEP, respectively). When the concentration of ascorbate is varied, inhibition with ACP appears to be better described by a pure noncompetitive pattern (Ki = Li = 1.6 mM), but inhibition with the acyclic analog AMEP is better described by a competitive pattern (Ki = 3.6 mM).
Fig. 1

Steady-state kinetic analysis using initial rate measurement velocities with nonlinear curve-fitting results for the inhibition of 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) by ACP with respect to ACC (top), HCO3 (middle), and ascorbate (bottom). The concentrations of ACP in the assays were 0 mM (filled circles), 0.5 mM (open squares), 1 mM (triangles), 2 mM (open circles), and 4 mM (filled squares). For the nonlinear curve-fitting of the kinetic data obtained when ACC was the substrate for which the concentration was varied, the experimental points at 4 mM ACP were not considered neither were the points filled in orange for 2 mM ACP

Table 1

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)

 

ACC

HCO3

Ascorbate

kcat (min−1)

11.3 ± 0.5

10.8 ± 0.2

11.9 ± 0.4

KM (mM)

0.15 ± 0.02

3.3 ± 0.4

2.7 ± 0.4

 

Inhibition type

Ki (mM)

Inhibition type

Ki (mM)

Inhibition type

Ki (mM)

ACP

C

1.5 ± 0.1

C

0.5 ± 0.1

NC

1.6 ± 0.4

AMEP

C

2.3 ± 0.4

C

1.6 ± 0.2

C

3.6 ± 0.4

When kept constant, the concentrations of the effectors were as follows: 1.25 mM ACC, 18 mM HCO3, 8 mM ascorbate, 40 μM Fe(II)

C competitive, NC pure noncompetitive (Ki = Li)

Determination of binding affinities by fluorescence quenching measurements

Intrinsic fluorescence of proteins, mainly of tryptophan residues, is a powerful tool to monitor substrate and cofactor binding [36]. This technique has been successfully used to study binding of metals to several proteins [46, 47]. It has also been used to study binding of cofactors to an ACCO-related enzyme, the 2-OG-dependent dioxygenase TfdA [48]. ACCO was purified as apoenzyme and the binding of the metal cofactor was investigated. The intrinsic fluorescence of tryptophans from ACCO (W31, W86, and W203) is characterized by an emission band at 345 nm (with λex = 290 nm) and was quenched upon iron binding. The addition of 200 μM Fe(II) under an inert atmosphere led to an approximately 30 % decrease of fluorescence intensity with almost no change in the emission maximum (344 nm). Fluorescence quenching upon addition of the metal cofactor was recorded under various conditions (see Fig. 2) and the data were analyzed using Eq. 6 to determine the dissociation constant for iron in the presence or absence of the other substrates/cofactors. The results are presented in Table 2. The dissociation constant for Fe(II) in the absence of any cofactor is 3.1 μM. In the presence of either ACC or bicarbonate at concentrations close to the standard conditions used in catalytic assays (1 and 18 mM, respectively), the dissociation constant for Fe(II) was not significantly modified. In the presence of both ACC and HCO3, the dissociation constant is 1.2 μM. This value is close to the value of 0.5 μM previously reported for tomato ACCO under catalytic conditions and, therefore, in presence of all substrates/cofactors required for activity [33].
Fig. 2

Changes in the fluorescence intensity under an inert atmosphere of 0.65 μM ACCO upon addition of left Fe(II) to apo-ACCO (squares) or apo-ACCO in the presence of 1 mM ACC and 18 mM bicarbonate (circles), and right ACC to apo-ACCO in the presence of 30 μM Fe(II) (squares) or apo-ACCO in the presence of 30 μM Fe(II) and 18 mM bicarbonate (circles). The data are normalized on the basis of the theoretical ΔImax derived from simulation

Table 2

Metal and 1-aminocyclopropane-1-carboxylic acid (ACC) dissociation constants determined by intrinsic fluorescence quenching using several conditions

Condition

Kd of Fe(II) (μM)

Kd of ACC (μM)

Apo-ACCO

3.1 ± 0.3

NQ

Apo-ACCO + 1 mM ACC

3.0 ± 0.5

Apo-ACCO + 18 mM HCO3

2.9 ± 0.5

NQ

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

NQ no significant quenching

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 [33].

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

We constructed a structure of tomato ACCO by homology modeling using the model of M. domestica ACCO as a template [8]. The modeled structure, compared with that of ACCO from P. hybrida, is displayed in Fig. 3. The global RMSD on backbone atoms between our model and the template is 0.17 Å with quasi-identical Ramachandran plots. The RMSD on backbone atoms between our model and the published structure of ACCO from P. hybrida is 5.94 Å, mainly accounting for the different conformation of the C-terminus.
Fig. 3

a Structure of ACCO from Petunia hybrida (Protein Data Bank code 1WA6). b Modeled structure obtained for tomato ACCO. The metal ion is displayed in yellow

Docking experiments: substrates

The modeled structure of ACCO from tomato was used to study the binding of ACC, HCO3, and ascorbate. Docking ACC to an ACCO/Fe enzyme led to a single cluster of solutions and ACC was found coordinated to the metal ion in a bidentate manner with the bound oxygen atom from ACC trans to D179 and the nitrogen atom in a quasiaxial position trans to H177 (see Fig. 4 and the electronic supplementary material). The Fe(II) ion is pentacoordinated. The structural index parameter can be calculated as defined by Addison et al. [49], and the value obtained, τ = 0.47, indicates a geometry intermediate between square-pyramidal and trigonal bipyramidal.1 A water molecule (or O2) could occupy the sixth position to provide a distorted octahedral geometry.
Fig. 4

Spatial arrangement at the active site of the modeled tomato ACCO obtained from docking experiments a of ACC, HCO3, and ascorbate and b of ACP, HCO3, and ascorbate. Residues involved in metal binding are displayed in orange and residues involved in binding HCO3 are displayed in green. The interactions between the enzyme and c ACC, HCO3, and ascorbate, and d ACP, HCO3, and ascorbate

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 [26], 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 [50]. 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.

Discussion

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 [8]. 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 [10], the R244 residue belonging to an RXS motif exclusively conserved in the structural subfamily of ACCO [12] 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. [9] 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 [53]. 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. [26] 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. [20] 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 [11]. 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. [27] 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 [58]. Similarly, it has been reported that mutations of residues R244 and S246 decrease the activity of the enzyme and increase KM for ACC [9]. In addition to this stabilization role, the twofold to 290-fold decrease of Vmax observed for those latter mutants [10] suggests that bicarbonate also plays an important role during catalysis, possibly assisting a proton transfer in the dioxygen activation pathway [27]. 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 [21] 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 [24]. 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. [9]. 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 [59]. 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. [30], 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 [60]. 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 [63], 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.

Footnotes

  1. 1.

    For square-pyramidal geometry τ = 0 and for trigonal bipyramidal geometry τ = 1.

  2. 2.

    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).

Notes

Acknowledgments

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.

Supplementary material

775_2012_910_MOESM1_ESM.pdf (267 kb)
Supplementary material 1 (PDF 266 kb)

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Copyright information

© SBIC 2012

Authors and Affiliations

  • Lydie Brisson
    • 1
  • Nadia El Bakkali-Taheri
    • 1
  • Michel Giorgi
    • 2
  • Antoine Fadel
    • 3
  • József Kaizer
    • 4
  • Marius Réglier
    • 1
  • Thierry Tron
    • 1
  • El Hassan Ajandouz
    • 1
  • A. Jalila Simaan
    • 1
  1. 1.Aix-Marseille Université and CNRS, Institut des Sciences Moléculaires de Marseille, UMR 7313Marseille Cedex 20France
  2. 2.SpectropoleAix-Marseille UniversitéMarseille Cedex 20France
  3. 3.Laboratoire de Synthèse Organique et Méthodologie, ICMMO, UMR 8182, Bât. 420Université Paris-SudOrsay CedexFrance
  4. 4.Department of ChemistryUniversity of PannoniaVeszprémHungary

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