Báo cáo khoa học: Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway ppt

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Báo cáo khoa học: Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway ppt

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Studies on structure–function relationships of indolepyruvate decarboxylase from Enterobacter cloacae , a key enzyme of the indole acetic acid pathway Anja Schu¨tz 1 , Ralph Golbik 1 , Kai Tittmann 1 , Dmitri I. Svergun 2,3 , Michel H. J. Koch 2 , Gerhard Hu¨ bner 1 and Stephan Ko¨ nig 1 1 Institut f € uur Biochemie, Fachbereich Biochemie/Biotechnologie, Martin-Luther-Universit € aat Halle-Wittenberg, Halle, Germany; 2 European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany; 3 Institute of Crystallography, Russian Academy of Sciences, Moscow, Russia Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofac- tors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoyl- formic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indica- tion for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem. 16, 1207–1216] and comparison with indole-3-pyruvic acid conversion by pyru- vate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase. Keywords: 1 benzoylformate; small angle X-ray scattering; steady-state kinetics; substrate specificity; thiamine diphosphate. The auxin indole-3-acetic acid, a phytohormone that promotes cell growth and elongation and influences rooting, is produced by plants [1,2] and plant-associated bacteria [3,4]. Both tryptophan-dependent and -independent path- ways of indole-3-acetic acid synthesis have been described [5,6]. Plants use several mechanisms to control levels of the active auxin indole-3-acetic acid. Thus, during different developmental stages, indole-3-acetic acid may originate from diverse sources for different auxin requirements, and under different environmental conditions. Bacteria primar- ily use tryptophan-dependent pathways. Phytopathogenic strains follow the indoleacetamide pathway and plant growth promoting strains the indolepyruvate pathway (Fig. 1). Indolepyruvate decarboxylase (IPDC), a key enzyme in the second pathway, is a thiamine diphosphate (ThDP)- and Mg 2+ -dependent homotetrameric enzyme that catalyses the decarboxylation of indole-3-pyruvate to indole-3-acetaldehyde [7–9]. Several microbial genes enco- ding IPDC have been reported, including one from Enterobacter cloacae isolated from the rhizosphere of actively growing cucumbers [10]. DNA sequence analyses revealed only one gene encoding EcIPDC. Its predicted amino acid sequence comprises 552 residues and has 40% identity to PDC from Kluyveromyces lactis (DCPY KLULA), 38% to PDC from Saccharomyces cerevisiae (DCP1 YEAST), and % 32% to PDC from Zea mays (DCP1 MAIZE), Oryza sativa (DCP1 ORYSA), Pisum sativum (DCP1 PEA), and to PDC from Zymomonas mobilis (DCPY ZYMO). In a previous study a molecular mass of 240 kDa was determined for the native state of EcIPDC, which corresponds to a tetramer with one type of subunit [7]. A sharp pH optimum in the catalytic activity of the enzyme assayed by quantitative HPLC was found at pH 6.4–6.6. The native substrate indolepyruvate has a low K m (15 l M ) in contrast with that of pyruvate (2.5 m M )[7]. Correspondence to S. Ko ¨ nig, Institut fu ¨ r Biochemie, Fachbereich Biochemie/Biotechnologie, Martin-Luther-Universita ¨ tHalle- Wittenberg, Kurt-Mothes-Str. 3, 06099 Halle/Saale, Germany. Fax: + 49 345 5527014, Tel.: + 49 345 5524829, E-mail: koenig@biochemtech.uni-halle.de Abbreviations: IDPC, indolepyruvate decarboxylase; EcIPDC, IPDC from Enterobacter cloacae;ScPDC,PDCfromSaccharomyces cerevisiae; ZmPDC, PDC from Zymomonas mobilis; ThDP, thiamine diphosphate. Enzymes: indolepyruvate decarboxylase (indole-3-pyruvate carboxy lyase; EC 4.1.1.74); pyruvate decarboxylase (2-oxoacid carboxy lyase; EC 4.1.1.1). Note:S 05 is the substrate concentration at half-maximum reaction rate for enzymes displaying cooperativity characterized by sigmoid reaction rate vs. substrate concentration plots. (Received 5 February 2003, revised 17 March 2003, accepted 2 April 2003) Eur. J. Biochem. 270, 2322–2331 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03602.x The pyruvate derivatives a-keto glutarate and b-phenyl- pyruvate inhibit EcIPDC activity. Indole and some similar metabolites such as L -tryptophan, indole-3-lactate, indole- 3-acetaldehyde, tryptophol, and indole-3-acetate have no effect on the enzymatic activity at a concentration of 0.5 m M [7]. Below, results on fast kinetics, substrate specificity, and cofactor binding of EcIPDC are presented. For the kinetic measurements a continuous optical assay was developed. The pH- and cofactor-dependent subunit association beha- viour was studied by small angle X-ray solution scattering. The catalytic specificities of EcIPDC, ScPDC, and ZmPDC for various substrates are discussed on the basis of their crystal structures. Materials and methods Reagents Horse liver alcohol dehydrogenase was from Roche Molecular Biochemicals Inc., yeast alcohol dehydrogenase and NADH were from Sigma-Aldrich Chemie GmbH. Unless otherwise stated all reagents were purchased from VWR International GmbH, Sigma-Aldrich Chemie GmbH, Carl Roth GmbH, and AppliChem GmbH. Bacterial strain and culture conditions The plasmid (3.8 kb) pIP362 expressed in the Escherichia coli strain JM109 (kindly provided by J. Koga, Meiji Seika Kaisha Ltd, Satima, Japan) encodes the gene isolated from E. cloacae [10]. A 6-L culture was grown for 24 h at 30 °C in media containing 2% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) sodium chloride, 0.1 m M thiamine, 0.1 m M magnesium sulphate, 0.01% (w/v) ampicillin, and 0.15 M potassium phosphate pH 6.5. Expression of the EcIPDC gene was induced by addition of 1 m M isopropyl thio-b- D -galactoside. Cells were harvested by centrifugation, quickly frozen in liquid nitrogen and stored at )80 °C. Protein purification About 25 g of cells were suspended in 40 mL 0.1 M potassium phosphate pH 6.5, containing 10 m M ThDP, Fig. 1. Scheme of the postulated biosynthesis pathway of indole-3-acetate from L -tryptophan in E. cloacae including the keto-enol tautomerism of indolepyruvate, modified according to Koga et al.[7].1, L -tryptophan aminotransferase; 2, indolelactate dehydrogenase; 3, indolepyruvate decarboxylase; 4, indoleacetaldehyde oxidase. Ó FEBS 2003 Structure–function studies of E. cloacae IDPC (Eur. J. Biochem. 270) 2323 10 m M magnesium sulphate, 1 m M EDTA, 5 m M dithio- threitol, and disrupted in a French press at 1200 bar (Gaulin, APV Homogeniser GmbH, Lu ¨ beck, Germany). The mixture was centrifuged at 70 000 g for 10 min and the pellet was discarded. Nucleic acids were precipitated by incubation with 0.1% (w/v) streptomycin sulphate for 45 min at 8 °C. A 15–30% (w/v) ammonium sulphate fractionation was performed at a protein concentration of 20 mgÆmL )1 . After centrifugation at 30 000 g for 5 min, the precipitate was dissolved in 20 mL 50 m M Mes/NaOH pH 6.5, containing 10 m M magnesium sulphate, 0.15 M ammonium sulphate and 1 m M dithiothreitol. The solution was applied to a Sephacryl S200HR column (5 · 95 cm, Amersham Biosciences) and eluted with the same buffer at 1mLÆmin )1 . The EcIPDC-containing fractions were pooled and concentrated by precipitation with ammonium sulphate (0.5 gÆmL )1 ). After centrifugation the precipitate was dissolved in 20 m M Mes/NaOH pH 6.5, 1 m M dithiothre- itol and this solution was desalted on a HiPrep desalting column (2.6 · 10 cm, Amersham Biosciences) and applied to a Source 15Q column (2.6 · 7 cm, Amersham Bio- sciences). Elution was performed using a linear gradient of 120 mL 0–25% 20 m M Mes/NaOH pH 6.5, 1 m M dithio- threitol, 0.25 M ammonium sulphate. The fractions with the highest catalytic activity and homogeneity were pooled, quickly frozen in liquid nitrogen after addition of 0.2 M ammonium sulphate, and stored at )80 °C. SDS/PAGE SDS/PAGE was carried out according to the method of Laemmli [11]. Gels (10% (w/v) acrylamide) were stained with Coomassie brillant blue G250. Determination of enzyme concentration The concentration of EcIPDC was determined spectro- photometrically at 280 nm using a calculated molecular absorption coefficient of 2 259 520 M )1 Æcm )1 [12]. ThDP- containing samples were analysed using the method of Bradford [13]. Syntheses of 4-substituted benzoylformates Syntheses were performed according to Hallmann and Ha ¨ gle [14] and Sultanov [15] by oxidation of the corres- ponding acetophenones by SeO 2 . Enzyme assays EcIPDC was preincubated with 15 m M ThDP/Mg 2+ pH 6.5 at room temperature for 20 min to saturate the enzyme with cofactors. Catalytic activities were measured using a coupled optical test [16,17] in 10 m M Mes pH 6.5, 0.2 m M NADH and two different alcohol dehydrogenases at 30 °C. Yeast alcohol dehydrogenase (15 UÆmL )1 )was used when the substrate was pyruvate, and horse liver alcohol dehydrogenase (1 UÆmL )1 ) was used with the substrates indolepyruvate, benzoylformate, and its 4-sub- stituted analogues. The decarboxylation of indolepyruvate, benzoylformate and its analogues was measured at 366 nm to reduce interference with the substrates that considerably absorb at 340 nm [17]. The conversion of pyruvate was followed at 340 nm. Indolepyruvate was preincubated in 10 m M Mes pH 6.5 at 25 °C for 45 min to ensure the generation of the ketone. The ability of ScPDC and ZmPDC to decarboxylate indolepyruvate was examined under the same conditions. In the case of ZmPDC maximum enzyme concentration was 2.3 mgÆmL )1 . Measurements with ScPDC were performed at an enzyme concentration of 90 lgÆmL )1 . The plots of the reaction rate vs. substrate concentration were fitted using the Michaelis–Menten equation in the case of EcIPDC, or according to a substrate activation mech- anism in the case of ScPDC [18]. For the substrate 4-NO 2 - benzoylformate the kinetic constants were estimated from the progress curves using the integrated Michaelis–Menten equation. Stopped-flow experiments were performed in 10 m M Mes pH 6.5, 0.55 m M NADH, 450 UÆmL )1 yeast alcohol dehy- drogenase and 25 m M pyruvate at 10 °Cand30°C. With 0.5 m M indolepyruvate and 20 m M benzoylformate 160 UÆmL )1 and 115 UÆmL )1 horse liver alcohol dehydro- genase were used, respectively 3 . For indolepyruvate the EcIPDC concentration was 0.3 mgÆmL )1 , for pyruvate it was 85 lgÆmL )1 , and for benzoylformate 3.5 lgÆmL )1 . The time-dependent inactivation of EcIPDC was exam- ined under various conditions using the coupled optical test with benzoylformate as substrate. Cofactor binding experiments were performed in 10 m M Mes pH 6.5, 50 m M Mg 2+ ,0.35m M NADH, 1 UÆmL )1 horse liver alcohol dehydrogenase, and 25 m M benzoyl- formate as substrate at 366 nm. To obtain the K d of the primary binding of ThDP the measurements were started with the apoenzyme–magnesium complex (10.7 lgÆmL )1 )at 20 °C. The progress curves were fitted according to Wang et al. [19] with an equation containing an exponential and a linear term. One unit of catalytic activity is defined as the amount of enzyme converting 1 lmol substrateÆmin )1 . 1 H NMR experiments on indolepyruvate To study the keto-enol tautomerism of indolepyruvate, 1 H NMR spectra of a solution of 1 m M indolepyruvate in 0.1 M potassium phosphate pH 6.7 [10% (v/v) D 2 O] were recorded 2–20 min after dissolving. Either presatu- ration, or watergate pulse programs were used to suppress the water signal. The chemical shifts refer to 3-(trimethylsilyl)-1-propane-sulphonate at 0 p.p.m. All experiments were performed on a Bruker ARX 500 Avance NMR spectrometer (proton frequency 500.13 MHz) at 20 °C. Determination of the molecular mass of EcIPDC Size exclusion chromatography. A Fractogel EMD Bio- SEC (S) column (2.6 · 70 cm, Merck KGaA) was equili- brated with 100 m M Mes pH 6.0 and 100 m M ammonium sulphate. EcIPDC was eluted with the same buffer at a flow rate of 1 mLÆmin )1 at 8 °C and detected by the protein absorbance at 280 nm. Ferritin (450 kDa), catalase (240 kDa), BSA (68 kDa), and ovalbumin (45 kDa) (Combithek, calibration proteins for chromatography, 2324 A. Schu ¨ tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Boehringer Mannheim GmbH) and ZmPDC (244 kDa) were used as molecular mass standards. Small angle X-ray solution scattering with synchrotron radiation. Data were collected on the X33 camera of the European Molecular Biology Laboratory outstation at Hasylab at the storage ring DORIS of the Deutsches Elektronen Synchrotron (DESY) in Hamburg [20–23]. Measurements were performed at a camera length of 1.9 m using multiwire proportional chambers with delay line readout [22] at a temperature of 12 °CandEcIPDC concentrations of about 5 mgÆmL )1 in 60 m M buffer at different pH values (citrate pH 5.6, Mes pH 6.1, BisTris pH 6.4, Pipes pH 6.8, Mops pH 7.2, Hepes pH 7.5, Tricine pH 8.1, Bicine pH 8.3, borate pH 9.2, Ches pH 9.5, and Caps pH 10.2), 62.5 m M ammonium sul- phate, 3 m M dithiothreitol in the presence or absence of 10 m M ThDP/Mg 2+ . The momentum transfer axis (s ¼ 4psinh/k,where2h is the scattering angle and k ¼ 0.15 nm, the X-ray wavelength) was calibrated using collagen or tripalmitin as standards. The scattering patterns were collected in 15 frames of 1 min to verify the absence of radiation damage. The experimental data was normalized to the intensity of the incident beam, corrected for the detector response, and buffer scattering was subtracted with propagation of statistical errors using the program SAPOKO (D.I.SvergunandM.H.J.Koch, unpublished data). To obtain the forward scattering intensity I 0 and the radius of gyration (R G ) the data was processed with the program GNOMOKO [24]. The molecular masses were calculated from the ratio of the forward scattering intensity of the samples and of the molecular mass standard BSA. The volume fractions of monomers, dimers and tetramers were determined using the program OLIGOMER (A. V. Sokolova, V. V. Volkov 4 and D. I. Svergun, unpublished data). All protein concentra- tions and pH values of the samples used for parameter calculation were determined after the measurements. Results Purification of EcIPDC The procedure, yielding the homogenous ThDP-free enzyme, comprises four steps: streptomycin sulphate treat- ment; ammonium sulphate precipitation; size exclusion chromatography; and anion exchange chromatography. After reconstitution of the holoenzyme the maximum specific activity was % 1UÆmg )1 using indolepyruvate as substrate. EcIPDC is quite stable at 40 °C without any further additions. A first-order rate constant of inactivation of 10 )5 Æs )1 was obtained in the elution buffer of the anion exchange chromatography. Ammonium sulphate (0.2 M ) stabilized the enzyme 14-fold. Further stabilization was achieved by addition of ThDP/Mg 2+ . Addition of 10% (v/v) glycerol had no effect. A molecular mass of 60 kDa per subunit was determined by SDS/PAGE, corresponding to the value calculated from the nucleotide sequence of the structural gene. The N-terminal amino acid sequence of the purified enzyme (Met-Arg-Thr-Pro-Tyr-Cys-Val-Ala) is identical to that of the nucleotide sequence of the EcIPDC gene (DCIP_ENTCL). Molecular mass determination and pH dependence of subunit association A molecular mass of 245 kDa corresponding to a tetramer was determined for EcIPDC at pH 6.0 by size exclusion chromatography and confirmed by small angle X-ray solution scattering with synchrotron radiation. Subunit association depends on pH. At pH values between 5.6 and 6.0 the tetrameric form of EcIPDC predominates (R G , 3.95– 4.1 nm; R G is the so-called radius of gyration, one of the structural parameters derived from a semi-logarithmic plot of scattering data according to Guinier [25]) followed by rapid dissociation into dimers at pH values between 6.7 and 7.4 (R G , 3.6–3.9 nm). At pH >8.0 R G values <3.1 nm indicate a predominant monomeric state of the enzyme. In the presence of cofactors the tetrameric holoenzyme is stabilized in the range pH 5.6–7.5. Data analysis with the program OLIGOMER demonstrated a pH-dependent equili- brium between tetramers and dimers at lower pH and dimers and monomers at higher pH. The presence of cofactors strongly suppressed significant accumulation of dimers (Fig. 2). 1 H NMR experiments on indolepyruvate EcIPDC is unable to decarboxylate freshly prepared solutions of indolepyruvate. Therefore, the chemical pro- perties and purity of indolepyruvate were characterized by 1 H NMR spectroscopy. The 1 H NMR spectrum of freshly dissolved indolepyruvate consists of the typical signals and spin systems of the indole moiety (triplets of 5-H and 6-H at 7.12 and 7.18 p.p.m., doublets of 4-H and 7-H at 7.43 and 7.75 p.p.m and the singlet of 2-H at 7.81 p.p.m. with identical integrals of all signals). The additional singlet of the pyruvyl moiety at 6.65 p.p.m. with a relative integral of 1 with respect to the indole protons is consistent with the occurrence of the enol form of indolepyruvate (Fig. 1). In the course of the establishment of the equilibrium % 85% of the enol form is converted into the ketone (half-time % 8 min at 20 °C) as deduced from the appearance of additional proton signals due to the indole part of Fig. 2. pH dependence of the oligomeric state of EcIPDC. Volume fractions were calculated from the scattering patterns with the program OLIGOMER in the absence of cofactors (A) and in the presence of 10 m M ThDP/Mg 2+ (B). (Circles and dotted lines, monomers; squares and full lines, dimers; triangles and dashed lines, tetramers; lines are drawn for better visualization only.) Ó FEBS 2003 Structure–function studies of E. cloacae IDPC (Eur. J. Biochem. 270) 2325 indolepyruvate (triplets of 5-H and 6-H at 7.04 and 7.12 p.p.m., doublets of 4-H and 7-H at 7.39 and 7.42 p.p.m and the singlet of 2-H at 7.16 p.p.m. with identical integrals of all signals) and to the b-CH 2 of the pyruvyl part (singlet at 4.15 p.p.m., relative integral of 2), respectively. As the ketone of indolepyruvate seems to be the true substrate species of EcIPDC catalysis, indolepyru- vate was always preincubated 45 min after dissolving to ensure the equilibrium between the tautomers. Steady state kinetics of EcIPDC In all previous kinetic studies on EcIPDC, a discontinuous assay based on HPLC was used [7]. To analyse the kinetic behaviour of the enzyme in more detail, a coupled optical assay was elaborated with alcohol dehydrogenase as auxiliary enzyme, catalysing the aldehyde–alcohol conver- sion similar to the assays established for pyruvate decarb- oxylase (PDC) and benzoylformate decarboxylase [16,17]. A rather low substrate specificity of the auxiliary enzyme horse liver alcohol dehydrogenase used in the latter assay and the high k cat /K m value (330 s )1 Æm M )1 ) for the substrate indole-3-acetaldehyde (data not shown) allowed application of this assay. Under all conditions used, the reaction rate is directly proportional to the EcIPDC concentration and independent of the concentration of the auxiliary enzyme, confirming that the coupled assay monitors the true rate of EcIPDC catalysis. Figs 3 and 4 and Table 1 illustrate the results of the steady-state kinetics for indolepyruvate, pyruvate, benzoylformate, and 4-substituted benzoylfor- mates (NO 2 -, Br-, Cl-, F-, C 2 H 5 -, CH 3 -, and CH 3 O-) as substrates of EcIPDC. The enzyme has the highest catalytic efficiency to the native substrate indolepyruvate, to 4-Cl- benzoylformate and to 4-Br-benzoylformate (k cat /K m >100 s )1 Æm M )1 ). The K m of these substrates is <50 l M . Benzoylformate has a rather low affinity to EcIPDC (K m 1.65 m M ), but its conversion resulted in the highest reaction rate. Compared to benzoylformate all substitutions of this substrate at the 4-position increase the affinity for the enzyme and decrease the turnover rate considerably (Table 1). The integrated Michaelis–Menten equation was used for the determination of the kinetic constants of 4-NO 2 -benzoylformate, the substrate with the lowest K m (5 ± 0.5 l M ) and a low k cat (0.4 ± 0.01 s )1 ). Pyruvate has Fig. 3. Dependence of the catalytic activity of EcIPDC on the concen- tration of substituted benzoylformates (Bf) measured in 10 m M Mes pH 6.5 at 30 °C. The lines represent the fits to hyperbolic kinetics. Fig. 4. Dependence of the catalytic activity of EcIPDC on the substrate concentration measured in 10 m M Mes pH 6.5 at 30 °C. The lines represent the fits to hyperbolic kinetics. Insets, corresponding stopped-flow progress curves. Straight lines are linear fits. Measurements were monitored at 340 nm for pyruvate and at 366 nm for the other substrates with a coupled optical test. Ipyr, indolepyruvate; Bf, benzoylformate; Pyr, pyruvate. 2326 A. Schu ¨ tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the lowest affinity of all substrates investigated (K m 3.38 m M ). The straight lines in the plots according to Hanes [26] (data not shown) demonstrate that there is no indication for any substrate activation processes in EcIPDC catalysis. The absence of lag phases in the progress curves obtained from stopped-flow experiments using indolepyruvate, pyruvate, and benzoylformate as substrates for EcIPDC at 30 °C (Fig. 4 insets) and 10 °C (data not shown) confirm these results. However, a weak substrate excess inhibition (K i 164 ± 16 m M ) was observed for pyruvate decarboxylation. Examination of the decarboxylation of indolepyruvate by ScPDC and ZmPDC The ability of ScPDC and ZmPDC to decarboxylate indolepyruvate was tested. In the case of ZmPDC no cata- lytic activity was found with indolepyruvate as substrate, even at very high enzyme concentrations (2.3 mgÆmL )1 ). However, ScPDC is able to convert indolepyruvate and displays, in contrast with EcIPDC, sigmoid kinetics as illustrated in Fig. 5. A k cat of 3.81 ± 0.24 s )1 andanS 0.5 - value of 0.7 m M was calculated according to the rate equation for substrate activation [18]. Cofactor binding experiments Cofactor binding was studied by restoration of the catalytic activity of the enzyme during reconstitution. Some progress curves are presented in Fig. 6. The pseudo first-order rate constants of reconstitution calculated from these time courses show a hyperbolic dependence on the ThDP concentration (at saturating Mg 2+ concentration), pointing to a two-step mechanism of cofactor binding (Fig. 6 inset) [27]. The calculated maximum rate constant of reconstitu- tion is % 0.03 s )1 and thus in the range of values determined for other PDCs ([28]; J. Scha ¨ ffner 5,6 , unpublished data; U. Mu ¨ cke 5,6 , unpublished data). A K d of 32.6 ± 4.6 l M determined for the binding of ThDP to EcIPDC is signifi- cantly lower than that of other PDCs except ZmPDC [29]. Discussion The purification procedure results in a homogenous ThDP- free enzyme that is stabilized by the addition of 0.2 M ammonium sulphate (inactivation rate constant 10 )6 s )1 at 40 °C) or cofactors ThDP and Mg 2+ .Kogaet al.[7]also described an effective stabilization of EcIPDC after addition of the cofactors. The enzyme is destabilized at low ionic strength. The stability of EcIPDC in aqueous solutions is higher than that of other PDCs. The rate constant of inactivation of PDC from Pisum sativum is about 10 )5 s )1 at 37 °C, that of ScPDC is one order of magnitude higher [30]. Table 1. Catalytic constants for the decarboxylation of different substrates by EcIPDC. The K m for indolepyruvate was calculated under consid- eration of the tautomer equilibrium (85% effective substrate concentration). k cat corresponds to the tetrameric enzyme, relative values to indolepyruvate. The kinetic constants result from hyperbolic fits to the reaction rate vs. substrate concentration plots. In the case of 4-NO 2 - benzoylformate values are obtained by fitting the progress curves using the integrated Michaelis–Menten equation (Ipyr, indolepyruvate, Pyr, pyruvate, Bf, benzoylformate). Substrates K m (l M ) K m (relative) k cat (s )1 ) k cat (relative) k cat /K m (s )1 Æm M )1 ) Ipyr 20 ± 1.3 1.0 3.9 ± 0.07 1.0 199 Pyr 3381 ± 179 169.1 3.5 ± 0.08 0.9 1 Bf 1646 ± 32 82.3 46.4 ± 1.23 11.9 28 4-NO 2 -Bf 5 ± 0.5 0.25 0.4 ± 0.01 0.1 80 4-Cl-Bf 48 ± 2.0 2.4 5.3 ± 0.05 1.4 110 4-Br-Bf 19 ± 1.0 0.95 3.2 ± 0.03 0.8 168 4-F-Bf 617 ± 32.0 30.9 22.7 ± 0.57 5.8 37 4-C 2 H 5 -Bf 111 ± 3.5 5.6 5.7 ± 0.06 1.5 51 4-CH 3 -Bf 127 ± 7.0 6.4 4.5 ± 0.08 1.2 35 4-CH 3 O-Bf 1043 ± 33.0 52.2 3.5 ± 0.09 0.9 3 Fig. 5. Dependence of the catalytic activity of ScPDC on the indole- pyruvate concentration. Measurements were carried out at 90 lgÆmL )1 ScPDC in 0.1 M Mes/NaOH pH 6.5 at 30 °Cand366nmwitha coupled optical test. (Circles, experimental data; solid line, fit accord- ing to the equation v([S]) ¼ V max Á½S 2 A þ BÁ½SþS 2 [18]). Ó FEBS 2003 Structure–function studies of E. cloacae IDPC (Eur. J. Biochem. 270) 2327 Stowe [31] postulated that indolepyruvate crystallizes in the enol form and is converted into the ketone at pH 8.0 and 25 °C within 20 min. Hydroxyphenylpyruvate and phenylpyruvate behave in a similar manner [32]. Schwarz and Bitancourt [33] demonstrated the tautomerism of indolepyruvate by TLC. Our time-dependent 1 HNMR measurements of aqueous solutions of indolepyruvate confirm these results. After 20 min incubation at 20 °C, 85% of the substrate is present as ketone. The remaining 15% is probably responsible for the formation of highly conjugated aromatic structures causing the well-known reddish discoloration of aqueous solutions of the substance. No EcIPDC activity is detectable with freshly prepared solutions of indolepyruvate as substrate, but maximum catalytic activity is obtained after incubation for about 45 min. Thus it can be concluded that only the ketone of indolepyruvate is the substrate for the enzyme. Application of a continuous optical assay for the steady- state measurements modified according to Weiss et al.[17] allowed detailed kinetic analysis of substrate specificity and cofactor binding of EcIPDC. The enzymatic conversion of all substrates studied in this work (pyruvate, the native substrate of PDC, benzoylformate, the native substrate of benzoylformate decarboxylase together with the 4-substi- tuted derivatives, indolepyruvate, the native substrate of IPDC) results in hyperbolic plots of catalytic activity vs. substrate concentration (Figs 3 and 4). Corresponding straight lines in Hanes plots (data not shown) and the absence of lag phases in the stopped-flow time courses (Fig. 4 insets) clearly demonstrates that there is no indica- tion for substrate activation behaviour in the EcIPDC catalysed reaction of the substrates indolepyruvate, pyru- vate, and benzoylformate. The same holds true for the ZmPDC catalysed reaction with pyruvate as substrate [34], but contrasts with all other PDCs exhibiting sigmoid dependencies in the plots of catalytic activity vs. substrate concentration [30,35–37]. The kinetic constants of EcIPDC summarized in Table 1 illustrate that indolepyruvate has the highest catalytic efficiency (k cat /K m ¼ 199 s )1 Æm M )1 ). Sur- prisingly, benzoylformate is converted more rapidly than thenativesubstrate(k cat 46.4 s )1 ), but it shows a K m value (1.65 m M ) about 80 times higher. In contrast, the kinetic constants of 4-Cl-benzoylformate and 4-Br-benzoylformate are comparable to that of the native substrate indolepyru- vate. Both halogenations seem to mimic the best substrate surrogates of indolepyruvate. The highest K M value and the lowest specificity are found for pyruvate and only this substrate displays a weak substrate excess inhibition (K i 164 m M ). The K m values determined for indolepyruvate and pyruvate correspond to those found by Koga et al.[7]using a discontinuous quantitative HPLC assay (15 l M and 2.5 m M , respectively). Interestingly, the K m value of pyru- vate in EcIPDC catalysis is similar to that found for all other PDCs and the same holds true for the weak substrate excess inhibition. However, the corresponding k cat value of EcIPDC is only about 2% of that of other PDCs. Hammett [38] developed a method to calculate the electronic effect of a substituent from studies on the dissociation of substituted benzoic acids in aqueous solu- tion. The corresponding constants are only of restricted value for other reactions. The modified substituent con- stants r p recommended by Hansch et al.[39]werefoundto be most suitable in the present case. The analysis of the kinetic constants of the 4-substituted benzoylformates as substrates for EcIPDC demonstrates that the dependence of the logarithm of k cat /k cat 0 vs. the substituent constant r p (Fig. 7) results in two linear plots with opposite slopes, one for the electron-donating substituents with a value of about 4.4, and one for the electron-withdrawing substituents with a value of about )2.5. This is indicative of an opposite effect of the electron-withdrawing and electron-donating substi- tuents on different rate-limiting steps in EcIPDC catalysis (formation of mandelyl-ThDP, decarboxylation or alde- hyde release), with a change in rate limiting step. To summarize, in EcIPDC catalysed reactions all substituents reduce the k cat value as compared with the unmodified benzoylformate; this is also the case for catalysis by benzoylformate decarboxylase from Pseudomonas putida [17]. However, in ScPDC [40,41] all benzoylformates with electron-withdrawing substituents exhibit a higher reaction rate and all benzoylformates with electron-donating sub- stituents have a lower one. EcIPDC binds all 4-substituted benzoylformates with a higher affinity than the unsubsti- tuted benzoylformate as is the case in ScPDC. With the exception of 4-methoxybenzoylformate the substituted benzoylformates have a lower affinity for benzoylformate decarboxylase than does benzoylformate itself. The hyperbolic dependence of the rate constants of reconstitution, calculated from the corresponding progress curves, on the concentration of ThDP (Fig. 6 inset) is indicative of a two-step mechanism of cofactor binding as Fig. 6. Progress curves of the reconstitution of EcIPDC with ThDP measured by restoration of the catalytic activity of the formed holo- enzyme for the substrate benzoylformate (25 m M )in10 m M Mes pH 6.5, 50 m M Mg 2+ ,0.35m M NADH, and 1 UÆmL -1 horse liver alcohol dehydrogenase at 20 °C. The reaction was started with EcIPDC (10.7 lgÆmL )1 ) at ThDP concentrations of 250, 120, 20, 12, 6, 3, 1.5, 1 and 0.5 l M (from left to right). Inset, dependence of the rate constant of reconstitution on the ThDP concentration, calculated from the progress curves. 2328 A. Schu ¨ tz et al. (Eur. J. Biochem. 270) Ó FEBS 2003 described previously by Schellenberger and Hu ¨ bner [27] and Eppendorfer et al. [42] for ScPDC. The reconstitution starts with binding of ThDP and Mg 2+ to the apoenzyme, followed by a conformational change to the catalytically active holoenzyme. Similar behaviour was found for ZmPDC (J. Scha ¨ ffner 7 , unpublished data), ScPDC [28] and PDC from Pisum sativum (U. Mu ¨ cke 8 , unpublished data). A resulting K d value of % 33 l M for the primary binding of ThDP to the enzyme saturated with magnesium ions illustrates a significantly higher affinity of the cofactor ThDPtoEcIPDCthantoScPDCandPDCfromPisum sativum (150–300 l M ). Even a higher affinity was found for ZmPDC [29]. A molecular mass corresponding to a tetramer of EcIPDC at pH 6.0 was determined by two independent methods, size exclusion chromatography and small angle X-ray solution scattering. These results suggest that the tetramer is stable in aqueous solution even without cofac- tors and that this oligomeric state is catalytically active in the presence of cofactors. Evaluation of the scattering experiments with ThDP-free EcIPDC demonstrates a pH- dependent equilibrium between tetramers, dimers and even monomers. A similar behaviour (without occurrence of a monomer fraction) was described for PDCs from various organisms, but not for ZmPDC, where the tetramer is stable from pH 5 to pH 9 [43]. The cofactors ThDP and Mg 2+ stabilize the tetrameric state of EcIPDC up to pH 7.5 (Fig. 2). A similar stabilization up to pH 8.5 was found for ScPDC [44]. The quality of the scattering patterns allowed the calculation of volume fractions of different oligomeric states of EcIPDC illustrating the pH-dependent subunit association equilibrium and demonstrating a further disso- ciation of EcIPDC into monomers at extreme alkaline pH values also described by Koga et al.[7]. As the crystal structure analysis of EcIPDC revealed some interesting similarities to other PDC species – a ScPDC-like open topology of the substrate binding site and a ZmPDC-like dimer assembly in the tetramer [45] – the conversion of indolepyruvate by those related PDCs was investigated. As expected from structural data [46,47], ZmPDC is not able to cleave indolepyruvate even at very high enzyme concentrations, whereas ScPDC decarboxylates indolepyruvate with a k cat of 3.81 ± 0.24 s )1 , a value similar to that of EcIPDC (3.9 ± 0.07 s )1 ).InthecaseofZmPDCthesizeofthe active site cavity is restricted by several amino acid changes [45,46]. In contrast, this bulky substrate fits into the active site of ScPDC and is decarboxylated. Differ- ences between the catalytic cleavage of indolepyruvate by ScPDC and EcIPDC can be found in the substrate affinity and in the reaction rate vs. substrate concentration plot. EcIPDC has a high affinity (K M 20 l M ) for indolepyruvate and follows Michaelis–Menten kinetics, whereas ScPDC exhibits sigmoid kinetics with a considerably lower affinity for the substrate (S 0.5 ¼ 0.7 m M ) (Fig. 5). The S 0.5 values for indolepyruvate and pyruvate (1.1 m M at pH 6.0; J. Ermer 9 , unpublished data) are in the same range for ScPDC. As in ZmPDC and plant PDCs prominent amino acid residues that restrict the size of the active site are conserved, such as Trp392 and Trp551 (ZmPDC numbering), one can assume that plant PDCs are also unable to accept indole- pyruvate as substrate. Consequently, other pathways for the biosynthesis of the phytohormone indoleacetic acid must exist, not excluding the existence of a specific plant IPDC. Yeast PDCs which do not possess such conserved space filling amino acid residues, have a more open topology of the substrate binding cavity and should thus presumably be capable of using indolepyruvate as substrate, although with a lower specificity than EcIPDC. In the active site, several amino acid residues assumed to play an important role in catalysis, such as Asp29, His115, His116, and Glu468 (EcIPDC numbering), are conserved in ZmPDC [48,49], ScPDC [50] and IPDCs suggesting a similar catalytic mechanism. 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