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Originally published In Press as doi:10.1074/jbc.M604477200 on June 12, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25076-25088, September 1, 2006
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Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

SPECTROSCOPIC AND KINETIC STUDIES*Formula

Vikrant M. Bhor{ddagger}, Sagarika Dev{ddagger}1, Ganga Ramu Vasanthakumar{ddagger}1, Parimal Kumar{ddagger}1, Sharmistha Sinha{ddagger}1, and Avadhesha Surolia{ddagger}§2

From the {ddagger}Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 and the §National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India

Received for publication, May 10, 2006 , and in revised form, June 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47 [EC] ), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the Mycobacterium tuberculosis KAPA synthase with both L- and D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k = 82.63 M–1 s–1) is ~5 times slower than that with L-alanine (k = 399.4 M–1 s–1). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed D-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k = 16.9 x 104 M–1 s–1). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, D-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (Ki = 114.83 µM) as well as 7,8-diaminopelargonic acid synthase (IC50 = 43.9 µM), the next enzyme of the pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Biotin (vitamin H) is an essential cofactor required for a number of carboxylation, transcarboxylation, and decarboxylation reactions in all living organisms. Its participation in gluconeogenesis, metabolism of amino acids, and initiation of fatty acid biosynthesis makes it an indispensable entity in cellular metabolism. However, only plants and microorganisms are capable of synthesizing biotin, whereas mammals obtain it either from the diet or from the intestinal microflora (1). The biotin biosynthesis pathway has been studied in detail in microbes like Escherichia coli, Bacillus subtilis, Bacillus sphaericus, and Saccharomyces cerevisiae and in plants like Arabidopsis thaliana and Lavandula vera. With the exception of the first step, which is the synthesis of pimeloyl-CoA, the remaining four steps of the pathway (Scheme 1) are invariant in all biotin-synthesizing organisms and are carried out by four committed enzymes (2). The first of these four steps is the decarboxylative condensation of pimeloyl-CoA and L-alanine to 7-keto-8-aminopelargonic acid (KAPA)3 catalyzed by KAPA synthase, followed by the DAPA synthase-catalyzed transfer of an amino group from S-adenosylmethionine to KAPA, forming DAPA. DAPA is then converted to dethiobiotin by the addition of CO2 in an ATP-dependent reaction catalyzed by dethiobiotin synthetase, and finally the biotin synthase-catalyzed insertion of a sulfur atom between the reactive methyl and methylene carbon atoms adjacent to the ureido ring of dethiobiotin to generate biotin (1–3).

KAPA synthase is a PLP-dependent enzyme belonging to subclass II of the aminotransferase family. Along with 5-aminolevulinate synthase, serine palmitoyltransferase, and 2-amino-3-oxobutyrate-CoA ligase, which typically catalyze the Claisen condensations between amino acids and acyl-CoA thioesters, it forms the {alpha}-oxoamine synthase subfamily. The similarities in the sequences of these enzymes as well as the reactions catalyzed by them indicate that they share a common catalytic mechanism (4–6). Availability of the three-dimensional structures of these enzymes has provided a structural basis for understanding the reactions catalyzed by them (4, 7–9). The knowledge of the reaction mechanism of KAPA synthase in particular is based on studies on the Bacillus sphaericus and E. coli enzymes (7, 10–11). It involves the displacement of lysine from the internal aldimine complex with PLP resulting in the formation of the external aldimine between PLP and L-alanine. This is followed by the abstraction of the C{alpha}-H proton of the L-alanine external aldimine by the conserved active site lysine, leading to the formation of a spectroscopically visible quinonoid intermediate, which attacks the thioester carbonyl of pimeloyl-CoA, releasing CoA and generating a beta-ketoacid aldimine intermediate. Decarboxylation of this intermediate yields the product quinonoid, which is protonated to form the product external aldimine, followed by the release of the product and reformation of the internal aldimine (7, 11).


Figure 1
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  		SCHEME 1
 
Although the chemical mechanism of KAPA synthase has been well characterized, the knowledge about the kinetic mechanism is still inadequate. KAPA synthase has been reported to show strict substrate stereospecificity such that it utilizes only L-alanine and not D-alanine (7, 12), which on the contrary acts as a competitive inhibitor for the B. sphaericus enzyme (12). We therefore undertook a comparative kinetic characterization of the interaction of the enzyme with L- and D-alanine with the purpose of understanding the kinetic basis for the substrate stereospecificity. The fact that no attempts have been made to characterize this enzyme from pathogenic bacteria prompted us to carry out these studies on the enzyme from Mycobacterium tuberculosis. We report here the rate constants for the formation of the reaction intermediates using pre-steady-state and steadystate kinetics. These studies also challenge the currently held view regarding the inhibitory action of D-alanine.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—L-Alanine, D-alanine, PLP, 5,5'-dithiolois-(2-nitrobenzoic acid) (DTNB), S-adenosylmethionine, 3-([1,1-dimethyl-2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, imidazole, kanamycin, beta-mercaptoethanol, and EDTA were obtained from Sigma. All other reagents used were of the highest available purity.

Cloning, Expression, and Purification of M. tuberculosis KAPA Synthase—The bioF gene (Rv1569) was amplified by PCR from the genomic DNA of M. tuberculosis H37Rv using the primers 5'-GAATTCCATATGAAAGCCGCCACGCAGG-3' and 5'-CCCAAGCTTCGGTGACGACCAGGATCGT-3'. The amplified products were cloned between the NdeI and Hind III sites in pET 28a+ vector (Novagen). Protein expression was carried out in E. coli BL21(DE3) cells (Novagen), and protein purification was carried out on nickel-nitrilotriacetic acid metal affinity resin (Novagen). The protein was eluted in 150 mM imidazole containing buffer (20 mM potassium phosphate, pH 7.7, 500 mM NaCl, 3 mM beta-mercaptoethanol, 15% glycerol, and 100 µM PLP) and was exchanged with imidazole-free buffer containing 20 mM potassium phosphate (pH 7.7), 150 mM NaCl, 3 mM beta-mercaptoethanol, 2 mM EDTA, 15% glycerol, and 100 µM PLP on a fast pressure liquid chromatography Sephadex G-25 column (Amersham Biosciences). The protein fractions so obtained were concentrated in Centricon concentrators (Amicon Ultra-15, Millipore), and the purity of the protein was judged by SDS-PAGE.

Synthesis of Pimeloyl-CoA, L-KAPA, and D-KAPA—Pimeloyl-CoA was synthesized by using mono-o-thiocresol pimelate and CoA according to the procedure of Kloss and Dickinson (13). L-KAPA was prepared as previously described by Nudelman et al. (14), starting from t-butoxycarbonyl-protected L-alanine. A slightly modified procedure was used to synthesize D-KAPA, starting from D-alanine as the chiral template. The measured optical rotations were opposite (for L-KAPA, [{alpha}]25D–4.32, c 0.5 in MeOH; for D-KAPA [{alpha}]25D + 4.28, c 0.5 in MeOH) in sign. For details see supplementary methods.

Spectrophotometric Determination of Kinetic Constants for M. tuberculosis KAPA Synthase—The activity of M. tuberculosis KAPA synthase was measured by a continuous spectrophotometric assay based on monitoring the release of CoASH from pimeloyl-CoA with Ellman's reagent, DTNB (15). A typical reaction consisted of 2.5 µM enzyme, 100 µM pimeloyl-CoA, 10 mM L-alanine, and 0.1 mM DTNB in 50 mM potassium phosphate buffer (pH 7.7). The reaction was initiated by addition of the enzyme, and absorbance changes at 412 nm due to the formation of the thiophenolate ({epsilon}412 = 13,600 M–1 cm–1) were monitored for 5 min at 25 °C in a V-530 UV-visible spectrophotometer (JASCO, Tokyo Japan). For determination of Km and kcat, concentrations of pimeloyl-CoA, L-alanine, and D-alanine were varied. Varying concentrations of D-alanine and D-KAPA were added to the standard reaction mix for determination of inhibitory constants. All the enzyme assays were carried out in triplicates. The initial velocity data were fitted to the Michaelis-Menten equation using nonlinear regression analysis by SigmaPlot 6.0. For determination of inhibitory constants, double reciprocal plots were plotted, and the data points were fitted using the appropriate equations (16).

Spectroscopic Characterization of M. tuberculosis KAPA Synthase—UV-visible spectra were recorded at 25 °C on a V-530 UV-visible spectrophotometer of the (a) enzyme alone, (b) the enzyme after addition of L-alanine (10 mM), (c) after addition of both L-alanine (10 mM) and pimeloyl-CoA (150 µM), and (d) after addition of the product, L-KAPA (4 mM). Ten different recordings were made in each of the abovementioned cases. The reaction buffer was 20 mM potassium phosphate (pH 7.7), 150 mM NaCl, 3 mM beta-mercaptoethanol, 2 mM EDTA, 15% glycerol, and 100 µM PLP. The enzyme concentration used was 25 µM.

Determination of Dissociation Constants for L- and D-Alanine— The reaction mix contained 25 µM enzyme and varying amounts of either L-alanine (0–20 mM) or D-alanine (0–80 mM). Pimeloyl-CoA (150 µM) was also added in a set of reactions with D-alanine. The reactants were allowed to equilibrate at 25 °C for 30 min after which spectra (300–600 nm) were recorded. The experiments were repeated five times. Changes in absorbance at 425 nm were plotted against L- or D-alanine concentrations, and the data points were fitted to a hyperbolic saturation curve (Equation 1),

Formula 1(Eq. 1)
where {Delta}Aobs is the observed change in absorbance, {Delta}Amax is the maximal absorbance change, [alanine] is the L- or D-alanine concentration, and Kd is the dissociation constant.

Determination of Pre-steady-state Rate Constants by Stopped-flow Spectroscopy—Fast reaction absorption kinetics experiments were performed on an Applied Photophysics SX.18MV stopped-flow spectrophotometer (dead time, 2 ms) maintained at a constant temperature by means of an external water bath. The concentrations of the reacting ligands were at least 10-fold greater than the enzyme concentration to ensure pseudo-first order reaction conditions, and the contents of the syringes were mixed in a ratio of 1:10 by volume. All measurements were performed in 20 mM potassium phosphate buffer (pH 7.7) containing 150 mM NaCl, 3 mM beta-mercaptoethanol, 2 mM EDTA, 15% glycerol, and 10 µM PLP. External aldimine formation of the enzyme (122 µM) with both L-alanine (5–90 mM) and D-alanine (25–150 mM) was monitored at 425 nm. Substrate-induced quinonoid formation was monitored at 532 nm, and the syringes contained enzyme (280 µM) and pimeloyl-CoA (100–280 µM). Product-induced quinonoid formation of the enzyme (245 µM) with L-KAPA and D-KAPA (30 mM each) was monitored at 500 nm. All traces are cumulative averages of at least 10 successive kinetic profiles. The data points were fitted to the monoexponential equation with a floating end point (Equation 2) to obtain the observed rate constants,

Formula 2(Eq. 2)
where a is a constant, b is the amplitude, and k is the observed rate constant. Curve fitting and analysis was done using an Acorn 5000, RISC workstation supplied by the manufacturer, and the quality of the fits was assessed by visual analyses of the calculated residuals. The dependence of the observed rate constants on the concentration of the reactants in excess was fit to Equation 3 for determination of the actual rate constants,

Formula 3(Eq. 3)
where kobs is the observed rate constant, kf and kr are the forward and reverse rate constants.

Determination of Steady-state Rate Constants—Substrate quinonoid formation with D-alanine was studied by monitoring the increase in absorbance at 532 nm in a V-530 UV-visible spectrophotometer at 25 °C for 2700 s, upon addition of pimeloyl-CoA (5–400 µM) to 30 µM enzyme premixed with 100 mM D-alanine. The data points were fit to monoexponential saturation curves to obtain the observed rate constants using Equation 2. Product quinonoid formation of the enzyme (30 µM) with either L-KAPA (1–80 mM) or D-KAPA (1–40 mM) was studied by monitoring the increase in absorbance at 500 nm for 2700 s. The data were fitted to Equation 4 to obtain the observed rate constants,

Formula 4(Eq. 4)
where A1 and A2 are the amplitudes of the two phases, y0 is a constant, and 1/t1 and 1/t2 are equal to the observed rate constants for the two phases. The plot of observed rate constants versus L-or D-KAPA concentration was fitted to Equation 5 to obtain the actual rate constants,

Formula 5(Eq. 5)
where kobs is the observed rate constant, kf and kr are the forward and reverse rate constants, and kd is the dissociation constant. In both the above cases, all the kinetic traces represent an average of at least ten different measurements.

Isothermal Titration Calorimetry—Determination of thermodynamic parameters and binding constants was carried out using a VP-Isothermal Titration Calorimeter (ITC) (MicroCal, Inc., Northampton, MA). All the titrations were performed thrice. Prior to measurements, the protein was dialyzed extensively against 20 mM potassium phosphate buffer (pH 7.7) containing 150 mM NaCl, 3 mM beta-mercaptoethanol, 2 mM EDTA, 15% glycerol, and 0.01 mM PLP, and the same buffer was used for preparation of the ligand solutions. Titrations were performed at 20 °C for studying the binding of the enzyme with (a) pimeloyl-CoA, by stepwise addition of a small volume (10 µl) of pimeloyl-CoA (1.5 mM) to the enzyme (120 µM) in the sample cell and (b) D-alanine, both in the presence and absence of pimeloyl-CoA. In this case, 20 µl of D-alanine (1.8 mM) was added stepwise to the enzyme (128 µM). For studying the binding of D-alanine to the enzyme in the presence of pimeloyl-CoA, the enzyme was allowed to equilibrate with pimeloyl-CoA (265 µM) in the sample cell for 30 min prior to titration. The raw calorimetric signals were integrated and corrected for the heat of dilution of pimeloyl-CoA and D-alanine. The resulting corrected binding isotherm was subjected to nonlinear least squares analysis using OriginTM 7.0 and fit to a single-site model to obtain the binding constant, Kb, the binding enthalpy, {Delta}H, and the stoichiometry of the interaction, n. The Gibbs free energy, {Delta}G, and the entropy, {Delta}S, were calculated using Equations 6 and 7.

Formula 6(Eq. 6)

Formula 7(Eq. 7)
Heat capacity changes associated with the formation of the enzyme-pimeloyl-CoA complex were determined by measuring the temperature dependence (15–30 °C) of the binding enthalpy. The relation of heat capacity change to the measured binding enthalpies and temperature is defined by Equation 8.

Formula 8(Eq. 8)
The linear least-squares fit of the dependence of the measured binding enthalpies on temperature provides {Delta}Cp, the heat change capacity for the binding process.

Mass Spectrometric Analyses—Liquid chromatography ESI-MS was carried out for characterization of the products of the enzyme-catalyzed reactions utilizing L- and D-alanine. An Esquire 3000 plus ion trap mass spectrometer (Bruker Daltonics) in positive ion mode was employed for this purpose. The reaction mix consisting of 10 mM L- or D-alanine, 100 µM pimeloyl-CoA, and 10 µM enzyme in 50 mM potassium phosphate buffer (pH 7.7) was incubated at 25 °C overnight. The reaction was terminated by heat inactivation of the enzyme followed by centrifugation at 15,000 rpm for 15 min. 10 µl of the supernatant was injected into a 5-µm Discovery C18 reversed-phase high-performance liquid chromatography column (Supelco), and separation of the reaction components was carried out at a constant formic acid concentration of 0.1% using a linear gradient of 5 to 95% acetonitrile in water at a flow rate of 0.2 ml/min over a period of 40 min. The total ion count in the range of 50–1800 m/z was recorded, and the molecular mass was determined using the Data Analysis software (3.1) (Bruker Daltonics).


Figure 2
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  		FIGURE 1.
Spectroscopic characterization of M. tuberculosis KAPA synthase. Representative spectra from ten individual absorption spectra recorded at 25 °C and pH 7.7 with 25 µM of the enzyme. A, absorption spectrum of the enzyme. B, formation of the external aldimine complex upon addition of 10 mM L-alanine to the enzyme. C, generation of a substrate quinonoid intermediate upon addition of pimeloyl-CoA (150 µM) to the preformed external aldimine complex shown in B. D, appearance of the product quinonoid intermediate after addition of L-KAPA (4 mM) to the enzyme.

 
M. tuberculosis DAPA Synthase Activity—The activity of the M. tuberculosis DAPA synthase recently characterized by us (17) was measured by the disc bioassay using the E. coli bioA109 (MEC1) strain (E. coli Genetic Stock Center, Yale University) as described by Eisenberg and Stoner (18). The reaction mixture consisted of L-KAPA (100 µM), S-adenosylmethionine (2.5 mM), and enzyme (5 µM) in 50mM 3-([1,1-dimethyl-2-hydroxyethyl] amino)-2-hydroxypropanesulfonic acid (pH 9.0) containing 150 mM NaCl, 5 mM beta-mercaptoethanol, 15% glycerol, and 10 µM PLP. The effect of D-KAPA on the enzyme activity was determined by adding it to the standard reaction mix. All the enzyme assays were carried out in triplicates.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization by Mass and Enzyme Activity
The M. tuberculosis gene Rv1569, annotated as the probable KAPA synthase was cloned and overexpressed, and the protein was purified to homogeneity by nickel-affinity chromatography. The molecular weight determined by SDS-PAGE was Mr 42,000, consistent with the ESI-MS analysis (Mr 42,248.59) and the molecular weight of Mr 42,194, calculated from the amino acid sequence. The recombinant protein exhibited activity in the enzyme assay, which is based on monitoring the release of CoA from pimeloyl-CoA by the Ellman's reagent, thereby confirming its identity as the M. tuberculosis KAPA synthase. The km values for L-alanine and pimeloyl-CoA are in the millimolar and micromolar range, respectively (Table 1), which is typical of acyl-CoA-condensing enzymes. However, the kinetic constants differ considerably from those of the E. coli, B. sphaericus (7), and the A. thaliana enzymes (19) (Table 1).


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  		TABLE 1
Steady-state rate constants for M. tuberculosis KAPA synthase

The enzyme assays were carried out in triplicates as described under "Experimental Procedures."

 
Spectroscopic Characterization of the Reaction Intermediates
The M. tuberculosis KAPA synthase exhibits spectroscopic properties characteristic of PLP-dependent enzymes with distinct absorbance maxima for various intermediates formed during the catalytic cycle. The absorbance maxima at 425 and 335 nm, which can be assigned to the two different forms of the enzyme-PLP internal aldimine complex (Fig. 1A) in which the C4'=N double bond is coplanar and non-coplanar, respectively, with the aromatic ring of PLP (7), indicates that the enzyme coexists in these two forms with the predominance of the non-coplanar form. Changes in absorption spectra were observed upon addition of both substrates and the product to the enzyme. Addition of L-alanine resulted in an increase in absorbance of the 425 nm band (Fig. 1B), indicating conversion of the internal to the external aldimine accompanied by a transition to the coplanar form. Addition of the other substrate, pimeloyl-CoA, to the external aldimine form of the enzyme, led to the appearance of a band with an absorption maximum at a higher wavelength, 532 nm, which is indicative of the formation of a quinonoid (carbanion) intermediate (Fig. 1C). Further, the addition of the product, L-KAPA, to the enzyme resulted in the appearance of an absorption band at 500 nm (Fig. 1D), most likely corresponding to the quinonoid form of the product-PLP complex. In contrast to the E. coli enzyme (7), the product-PLP quinonoid of M. tuberculosis KAPA synthase absorbs at a wavelength shorter than the substrate-PLP quinonoid intermediate.

Interaction with Pimeloyl-CoA
The binding of pimeloyl-CoA to M. tuberculosis KAPA synthase was studied by ITC, because this interaction does not involve any spectroscopic changes. A typical ITC profile for the binding reaction at 20 °C is shown in Fig. 2A, and the temperature dependence of the thermodynamic parameters for the pimeloyl-CoA-enzyme interactions are given in Table 2. The binding of pimeloyl-CoA to the enzyme occurs at a single site with high affinity as indicated by the values of binding constants (Kb = (0.51 ± 0.023) x 106 M–1). The binding also exhibits a temperature-dependent enthalpy-entropy compensation, which leads to small changes in binding affinity with change in temperature. The heat capacity change, {Delta}Cp, for the binding reaction was –332.34 cal/mol/K (Fig. 2B).


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  		TABLE 2
Thermodynamic parameters governing binding of pimeloyl-CoA to M. tuberculosis KAPA synthase

Titrations were carried out in 20 mM potassium phosphate buffer (pH 7.7) containing 20% glycerol, 150 mM NaCl, 2 mM EDTA, 3 mM beta-mercaptoethanol, and 10 µM PLP using a VP-ITC calorimeter (Microcal, Inc., Northampton, MA). Kb and {Delta}H were determined by fitting the ITC data to a single-site binding model in Origin software, whereas {Delta}G and {Delta}S were calculated using the equations, {Delta}G = RT ln Kb and {Delta}G = {Delta}H — T{Delta}S. The values given below represent results of a single titration, and in multiple titrations the values of Kb and {Delta}H are reproducible within 2-fold and 1 kcal/mol, respectively.

 


Figure 3
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  		FIGURE 2.
Thermodynamic characterization of the interaction of pimeloyl-CoA with M. tuberculosis KAPA synthase. A, upper panel shows a representative ITC profile depicting the heat exchanged during the titration of the enzyme (120µM) with 10-µl volumes of pimeloyl-CoA (1.5 mM) in 20 mM potassium phosphate buffer (pH 7.7) containing 150 mM NaCl, 3 mM beta-mercaptoethanol, 2 mM EDTA, 15% glycerol, and 0.01 mM PLP at 20 °C. The lower panel shows the least squares fit of the data in the upper panel to obtain the binding constant, Kb = (0.51 ± 0.023) x 106 M–1 as well as the binding enthalpy, {Delta}H = –5.30 ± 0.025 kcal/mol. B, the plot of the temperature dependence of the binding enthalpy gave the heat change capacity, {Delta}Cp = –332.34 cal/mol/K, associated with the binding of pimeloyl-CoA.

 
Interaction with L- and D-Alanine
Addition of both L- and D-alanine to the enzyme resulted in the formation of an external aldimine, evidenced by an increase in absorbance at 425 nm. Plots of the increase in absorbance at 425 nm against alanine concentrations yielded hyperbolic curves (Fig. 3, A and B) with dissociation constants, Kd of 1.93 ± 0.28 mM and 12.49 ± 1.80 mM for L- and D-alanine, respectively. These values are in agreement with those obtained for the corresponding E. coli (7) and B. sphaericus enzymes (12), which also exhibit an increased affinity for D-alanine in the presence of pimeloyl-CoA. However, in contrast to both these enzymes (7, 12), addition of increasing concentrations of D-alanine to M. tuberculosis KAPA synthase in the presence of saturating concentrations of pimeloyl-CoA, resulted in the appearance of an absorption band centered at 532 nm, apart from the formation of external aldimine (Fig. 3C). This indicates the formation of a quinonoid intermediate upon interaction of pimeloyl-CoA with the external aldimine between the enzyme and D-alanine. The increase in affinity of D-alanine for the enzyme in the presence of pimeloyl-CoA (7, 12) was confirmed by measuring the binding constants for D-alanine in the absence (Kb = (9.41 ± 1.24) x 103 M–1) (Fig. 3D) and in the presence of pimeloyl-CoA (Kb = (21.7 ± 6.31) x 103 M–1) (Fig. 3E) using ITC, a technique for unambiguous determination of binding constants, which unlike spectroscopic measurements does not involve an incubation period and therefore does not allow appreciable quinonoid intermediate formation to occur. The binding of L-alanine to the enzyme, however, could not be studied by ITC because no appreciable heat change was observed in this case.

Pre-steady-state Kinetic Characterization of the Reaction Intermediates
The difference in the interaction of the enzyme with L- and D-alanine was further probed by determination of pre-steadystate rate constants for external aldimine (425 nm) and substrate quinonoid (532 nm) formation with each enantiomer. Further, a comparison of the rate of product quinonoid (500 nm) formation by the interaction of the enzyme with its physiological product, L-KAPA, and its enantiomer, D-KAPA, the product most likely to be formed by the reaction between D-alanine and pimeloyl-CoA was also carried out.

External Aldimine—The kinetic trace for external aldimine formation with L-alanine exhibited a simple monoexponential increase (Fig. 4A), the fit of which gave observed rate constants that upon plotting against L-alanine concentration gave the second order rate constant, k1, 399.4 ± 30.29 M–1 s–1 (Fig. 4A), 50 times lower than that for the E. coli enzyme (7). The reverse rate constant was 23.43 ± 1.44 s–1. The kinetic trace obtained with D-alanine also exhibited a monoexponential rise. Plotting the concentration dependence of the observed rate constants yielded a second-order rate constant, k1 = 82.63 ± 5.66 M–1 s–1 (Fig. 4B), ~5 times slower than the formation of the external aldimine with L-alanine. The reverse rate constant was 15.4 ± 0.47 s–1.

Substrate Quinonoid—The kinetic trace of substrate quinonoid formation with pimeloyl-CoA and L-alanine, exhibited two distinct phases (Fig. 4C). One lag or spectroscopically silent phase was observed up to 30 ms. This phase was found to be independent of temperature (10–40 °C) and concentration of pimeloyl-CoA (100–300 µM). The lag phase was followed by a single exponential rise with a rate constant of (16.9 ± 1.54) x 104 M–1 s–1. The reverse rate constant was 79.25 ± 3.08 s–1. In the corresponding reaction between D-alanine and pimeloyl-CoA, no appreciable increase in absorbance was seen even up to 10 s, indicating the absence of a faster phase. Thus as compared with L-alanine, the formation of substrate quinonoid intermediate is much slower in the case of D-alanine.

Product Quinonoid—The time course for the reaction between L-KAPA and the enzyme (Fig. 5A), fits to a single exponential with a rate of 145.6 ± 8.3 s–1. The pattern for the reaction with D-KAPA (Fig. 5B) also fits to a single exponential rise but with a rate of 27.45 ± 0.62 s–1, considerably slower than that for L-KAPA. The low signal-to-noise ratios due to small absorbance changes prevented an accurate determination of the second order rate constants.

Steady-state Kinetic Characterization
The absence of a fast reaction phase in the formation of the substrate quinonoid with D-alanine and pimeloyl-CoA implies slower reaction kinetics. The reaction was therefore most appropriately studied by steady-state kinetics. The kinetics, however appears to be complex with the presence of two phases (Fig. 4C), both of which showed single exponential increase with rate constants, 6.42 ± 0.30 x 10–3 s–1 and 1.2 ± 0.1 x 10–3 s–1 for the first and the second phases, respectively. Steady-state kinetics was also employed to further probe the interaction between the enzyme and L- as well as D-KAPA. The time course of the reactions between L-KAPA and the enzyme (Fig. 5C), as well as between D-KAPA and the enzyme (Fig. 5D), can be best described by biphasic processes. The rate dependence on L-KAPA concentration was determined (Fig. 5C, inset). The fast phase was saturable, and a fit to Equation 5 gave values of Kd = 5.40 ± 2.72 mM, kf = 39.3 ± 2.5 M–1 s–1, and kr = 0.01 ± 0.004 s–1. The rate dependence on D-KAPA concentration (Fig. 5D, inset) was also determined and the fast phase was fit to Equation 3 to obtain the rate constants, kf = 1.03 ± 0.07 M–1 s–1 and kr = 13.0 ± 0.70 x 10–3 s–1. The second slow phase for both L-KAPA and D-KAPA was concentration-dependent, indicating that it is associated with the isomerization of the enzyme-L-KAPA and the enzyme-D-KAPA complex.


Figure 4
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  		FIGURE 3.
Binding of L- and D-alanine to M. tuberculosis KAPA synthase. Representative spectra from five individual absorption spectra acquired at 25 °C and pH 7.7 with 25 µM of the enzyme. A, the enzyme was pre-equilibrated with L-alanine (0–20 mM, from bottom to top), and the spectra were recorded. Data were fitted to a hyperbolic saturation curve as shown in the inset to obtain the dissociation constant, Kd = 1.93 ± 0.28 mM. B, pre-equilibration of the enzyme with D-alanine (0–80 mM, from bottom to top) was followed by recording the spectra and the fitting of the data to a hyperbolic curve gave the dissociation constant, Kd = 12.49 ± 1.80 mM. C, addition of pimeloyl-CoA (150 µM) to the preformed external aldimine complex with D-alanine as shown in B. Calorimetric titration of the binding of D-alanine (1.8 mM) to the enzyme (128 µM)(D) in the absence and E, in the presence of pimeloyl-CoA (265 µM) yielded the binding constants, (9.41 ± 1.24) x 103 M–1 and (21.7 ± 6.31) x 103 M–1. The titrations were performed thrice, and the figures are representative of the results of a single titration.

 


Figure 5
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  		FIGURE 4.
Kinetic characterization of the external aldimine and substrate quinonoid intermediates of M. tuberculosis KAPA synthase. The time course for the formation of the reaction intermediates was monitored at 25 °C and pH 7.7. All the traces shown are cumulative averages of at least ten successive kinetic profiles. A, pre-steady-state kinetic trace of external aldimine formation with L-alanine (20 mM) monitored at 425 nm. The plot of the observed rate against concentration shown in the inset, gave the rate constant k1 = 399.4 ± 30.29 M–1 s–1. B, pre-steady-state kinetic trace of external aldimine formation with D-alanine (50 mM) monitored at 425 nm. The plot of the observed rate against concentration shown in the inset, gave the rate constant k1 = 82.63 ± 5.66 M–1 s–1. C, pre-steady-state kinetic trace of substrate quinonoid formation with L-alanine (10 mM) and pimeloyl-CoA (280 µM) monitored at 532 nm. The plot of the observed rate against concentration shown in the inset, gave the rate constant k1 = (16.9 ± 1.54) x 104 M–1 s–1. D, steady-state kinetic trace of the substrate quinonoid formation with D-alanine (100 mM) and pimeloyl-CoA (400 µM) monitored at 532 nm. The two phases were separately fitted to Equation 2 to get the rate constants, (6.42 ± 0.30) x 10–3 s–1 and (1.2 ± 0.1) x 10–3 s–1, for the first and the second phases, respectively.

 
Mass Spectrometric Analyses of the Reaction Products
Mass spectrometric analyses of the reaction mixtures of the enzyme-catalyzed reaction between L-alanine and pimeloyl-CoA, and D-alanine and pimeloyl-CoA, showed the presence of peaks corresponding to masses of 210.7 and 211.1 Da (Fig. 6), respectively, representing the sodium adduct of the reaction product, KAPA (188 Da).

Inhibition of M. tuberculosis KAPA Synthase
The D-enantiomer of alanine was also utilized by the M. tuberculosis KAPA synthase as a substrate and its Km and kcat values are reported in Table 1. However, addition of D-alanine to the standard enzyme reaction containing L-alanine and pimeloyl-CoA resulted in linear mixed type inhibition of the enzyme. The Ki was found to be 464.78 ± 32.76 µM (Fig. 7A). Similarly, the D-enantiomer of the product KAPA also brought about a linear mixed type inhibition of the enzyme with a Ki = 114.83 ± 5.74 µM (Fig. 7B) in contrast to the physiological product of the reaction, the L-enantiomer, which did not inhibit the reaction even up to a concentration of 2 mM. The Km for D-alanine was found to be lower than that for L-alanine in contrast to the results of the spectroscopic measurements, which indicate that L-alanine binds to the enzyme with higher affinity as compared with D-alanine. The fact, that the reaction product of the D-alanine-utilizing reaction, D-KAPA, inhibits the enzyme, may interfere with the accurate determination of Km for D-alanine and could therefore explain the observed discrepancy between the results for D-alanine obtained by the enzyme assay and spectroscopic measurements.


Figure 6
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  		FIGURE 5.
Kinetic characterization of the product quinonoid intermediate of M. tuberculosis KAPA synthase. The time course for the formation of the product quinonoid was monitored at 25 °C and pH 7.7. All traces represent averages of at least ten successive kinetic profiles. A, pre-steady-state kinetic trace with L-KAPA (30 mM) monitored at 500 nm. Fitting the data to a monoexponential increase gave a rate of 145.6 ± 8.3 s–1. B, pre-steady-state kinetic trace with D-KAPA (30 mM) monitored at 500 nm. Fitting the data to a monoexponential increase gave a rate of 27.45 ± 0.62 s–1. C, steady-state kinetic trace of the product quinonoid formation with L-KAPA (5 mM) monitored at 500 nm in a bi-exponential process. The concentration dependence of the two phases is shown in the inset. The fast phase was fit to Equation 5 and gave values of Kd, kf, and kr of 5.40 ± 2.72 mM, 39.3 ± 2.5 M–1 s–1, and 0.01 ± 0.004 s–1, respectively. The slow phase was concentration-independent. D, steady-state kinetic trace of product quinonoid formation with D-KAPA (5 mM) monitored at 500 nm in a biexponential process. The concentration dependence of the two phases is shown in the inset. The fast phase was fit to Equation 3 and gave values of kf = 1.03 ± 0.07 M–1 s–1 and kr = 13.0 ± 0.7 x 10–3 s–1. The slow phase was concentration-independent.

 
Inhibition of M. tuberculosis DAPA Synthase
The D-enantiomer of KAPA exhibited negligible activity when used as a substrate for DAPA synthase as compared with the natural substrate, L-KAPA. However, D-KAPA brought about inhibition of DAPA synthase activity, and the IC50 was found to be 43.9 ± 8.5 µM (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Tuberculosis continues to be a major global health concern even in the 21st century claiming millions of lives every year. The lack of a uniformly effective vaccine and the existence of multidrug-resistant strains of the causative agent, M. tuberculosis, necessitate a search for unexplored targets for drug development (20). Despite the fact that enzymes involved in biotin biosynthesis are not present in humans (1–3), none of the studies so far have attempted to characterize these enzymes from pathogenic bacteria with the objective of rational development of inhibitors. We report the characterization of M. tuberculosis KAPA synthase, the enzyme catalyzing the first committed step in the biotin biosynthesis pathway.

The steady-state spectrum represents an intermediate whose formation is rate-limiting, or it represents the sum of more than one intermediate if multiple steps are partially rate-limiting (21), and therefore the presence of both the external aldimine as well as the substrate quinonoid in the steady-state spectrum (Fig. 1C) indicates that the formation of these intermediates may be partially rate-determining. Thermodynamic characterization of the enzyme by ITC, the first for any member of the biotin biosynthesis pathway, proves unequivocally that pimeloyl-CoA interacts with the enzyme even in the absence of L-alanine and thereby suggests that substrate binding could also occur in a random rather than a strictly sequential order. Further, the binding involves hydrophobic interactions presumably between the acyl chain of the pimelate group and hydrophobic residues in the enzyme as indicated by the large negative heat capacity change associated with its binding.


Figure 7
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  		FIGURE 6.
Mass spectrometric analyses of the reaction products of M. tuberculosis KAPA synthase. The reaction mix consisting of either L-or D-alanine (10 mM), pimeloyl-CoA (100 µM), and enzyme (10 µM) in 50 mM potassium phosphate buffer (pH 7.7) was incubated at 25 °C overnight, and the reaction was terminated by heat inactivation. A, LC profile and B, ESI-MS profile of the reaction utilizing L-alanine shows the presence of a peak corresponding to a mass of 210.7 Da. C, LC profile and D, ESI-MS profile of the product of the reaction utilizing D-alanine shows the presence of a peak corresponding to a mass of 211.1 Da. The masses of both these peaks correspond to the sodium adduct of KAPA (188 Da).

 
The first step in the catalytic cycle is the conversion of the internal aldimine to the external aldimine. The pre-steadystate kinetic trace of external aldimine formation with L-alanine reflects the formation of geminal diamine intermediates as well as the conformational transition of the cofactor, i.e. a rotation around the C4–C4' bond to orient the plane determined by the lysine nitrogen, C4' and C4, perpendicular to the pyridine ring to facilitate the preferential expulsion of the lysine amino group (22) over the amino group of L-alanine. The second-order rate constants for external aldimine formation with both L- and D-alanine are several orders of magnitude slower than those expected for a diffusion-controlled reaction. This suggests that the formation of the external aldimine complex may be preceded by the rapid formation of a diffusion-controlled reaction intermediate complex as depicted in Reaction 1,

Formula 9REACTION 1
where K1 = k1/k–1 and KII = k2/k–2 are the association constants for the two steps (23, 24, 25), E is the enzyme, S is the ligand i.e. alanine, and ESi and ES represent the intermediate and the final external aldimine complexes, respectively.

However, the failure to observe any intermediate could be due to one or more of the following reasons: (a) the formation of the intermediate is too rapid to be observed and may occur within the dead time of the stopped-flow instrument; (b) the formation of the reaction intermediate occurs without any observable change in absorbance; or (c) the dissociation constant, 1/K1, in case of the intermediate formation is too high to allow significant quantities of the intermediate to accumulate during the course of the reaction (23, 26).

Alternatively, the formation of the external aldimine could be described as a single-step bimolecular reaction and may proceed without the occurrence of a reaction intermediate as given in the following reaction scheme (Reaction 2).

Formula 10REACTION 2
In this case steric factors may be responsible for the low second order reaction rates (23, 27).

Although the enzyme has a lower affinity for D-alanine, it is still able to form an external aldimine with it ~5 times slower than L-alanine. Interestingly, the formation of the external aldimine with D-alanine seems to be more favored in case of the M. tuberculosis enzyme than the E. coli enzyme, where external aldimine formation with D-alanine is 160 times slower than with L-alanine (7). The reason for the slower kinetics of external aldimine formation with D-alanine, or the fact that it is more favored in the case of the M. tuberculosis enzyme compared with its E. coli counterpart, is not apparent at present but could be caused by some unfavorable contacts arising due to inversion of stereochemistry, which probably also limits the change to a predominantly coplanar form.


Figure 8
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  		FIGURE 7.
Inhibition of M. tuberculosis KAPA synthase and DAPA synthase. A, Lineweaver-Burk plot showing linear mixed type inhibition of the M. tuberculosis KAPA synthase by D-alanine ({blacksquare}) 0 mM, ({blacksquare}) 0.5 mM, ({blacktriangleup}) 1 mM, and ({square}) 2 mM. Inset shows the slope replot of the Lineweaver-Burk plot. Ki = 464.78 ± 32.76 µM. B, Lineweaver-Burk plot showing linear mixed type inhibition of the M. tuberculosis KAPA synthase by D-KAPA ({blacksquare}) 0 mM, ({triangledown}) 0.125 mM, ({blacktriangledown}) 0.25 mM, and ({square}) 0.33 mM. Inset shows the slope replot of the Lineweaver-Burk plot. Ki = 114.83 ± 5.74 µM. C, inhibition of M. tuberculosis DAPA synthase by D-KAPA. Plot of percent activity of DAPA synthase against concentration of D-KAPA gave an IC50 value of 43.9 ± 8.5 µM. All the values are averages of three different experiments.

 
The next event in the catalytic cycle is the formation of the substrate quinonoid intermediate. The 30-ms lag phase observed prior to quinonoid formation most likely corresponds to the conformational transitions of the pyridine ring of PLP like the rotation around the N–C{alpha} bond induced by the binding of pimeloyl-CoA. This will result in the placement of the C{alpha} of L-alanine close to the conserved lysine to facilitate rapid proton abstraction and the consequent generation of a resonance-stabilized quinonoid intermediate, as suggested in the case of the E. coli enzyme (7). The dramatic increase in the rate of substrate quinonoid formation compared with external aldimine formation implies the acceleration of the conformational changes necessary to facilitate proton abstraction, upon binding of pimeloyl-CoA.

Surprisingly, in addition to forming external aldimine, D-alanine was also able to form the substrate quinonoid intermediate. The formation of the quinonoid intermediate of M. tuberculosis KAPA synthase with D-alanine is intriguing, because such an intermediate has not been observed previously with its counterparts from E. coli (7) and B. sphaericus (12). The absence of a fast reaction phase suggests the occurrence of a longer lag period, which is most likely the time required for the conformational transitions of the pyridine ring to facilitate the abstraction of the C{alpha} proton of D-alanine. The presence of some unfavorable contacts that delay the attainment of a planar geometry required to position the C{alpha} atom close to the active site lysine for proton abstraction may be responsible for the slower kinetics. Although the formation of external aldimine by D-alanine is only 5 times slower than L-alanine, the formation of substrate quinonoid by D-alanine appears to be much slower than the corresponding event with L-alanine. The decrease in the dissociation constant of D-alanine in the presence of pimeloyl-CoA supports the idea that binding of pimeloyl-CoA blocks the exit of D-alanine (7) and thereby promotes the slow processes of formation of the external aldimine and its progression to quinonoid.

Although the formation of the quinonoid intermediate with D-alanine clearly suggests that D-alanine can act as a substrate for M. tuberculosis KAPA synthase, the fact that the enzyme exhibits activity in the spectrophotometric assay based on the measurement of CoA release, using D-alanine and pimeloyl-CoA, constitutes additional proof of its ability to utilize D-alanine as a substrate. The finding that the products of the reaction with both D-alanine and L-alanine have a mass identical to KAPA, further confirms the fact that D-alanine acts as a substrate for the M. tuberculosis KAPA synthase. Analogous to L-KAPA being the product of the enzyme reaction utilizing L-alanine, the product of the enzyme-catalyzed reaction utilizing D-alanine is the D-enantiomer of KAPA i.e. D-KAPA.

Most enzymes exhibit strict stereospecificity with respect to utilization of substrates. However, there are exceptions wherein enzymes display a broad substrate stereospecificity such that they can bind as well as process both enantiomers of their respective substrates (28–32). Additionally, some enzymes from different sources exhibit differences in their substrate stereospecificities. Tryptophan 2,3-dioxygenase from mammalian (rat, mouse, rabbit, and human) liver utilizes both L- and D-isomers of tryptophan, whereas the same enzyme from Pseudomonas fluorescens utilizes only the L-isomer (28). The utilization of both L- and D-alanine as substrates by the M. tuberculosis KAPA synthase demonstrates its broad substrate stereospecificity. PLP-dependent enzymes exhibit stereospecificity at the level of the exchange of {alpha}-protons of their amino acid substrates by catalyzing this reaction either on the si- (sinister) or the re- (rectus) face of the planar external aldimine intermediate (29, 32). The ability of the M. tuberculosis KAPA synthase to exchange{alpha}-protons of both L- and D-enantiomers on both the si- and the re-faces as reported in case of the PLP-dependent amino acid racemases (32) may give rise to its broad stereospecificity.

Contrary to our finding of D-alanine being a substrate for the M. tuberculosis KAPA synthase, it has previously been reported to act as a competitive inhibitor for the B. sphaericus enzyme (12). Indeed, addition of D-alanine resulted in inhibition of the M. tuberculosis KAPA synthase-catalyzed reaction between L-alanine and pimeloyl-CoA. However, the inhibition was of linear mixed type instead of competitive. The fact that D-KAPA, the enantiomer of the physiological product of the reaction, L-KAPA, also brings about a linear mixed type inhibition of the enzyme akin to D-alanine, hints toward a plausible explanation for the apparently paradoxical behavior of D-alanine. Linear mixed type inhibition is characterized by the binding of the substrate and the inhibitor, independently and reversibly to the enzyme without competing for a common site resulting in the formation of ES, EI, and IES complexes, and the IES complex is catalytically inactive (16). Both, D-KAPA and the two enantiomers of alanine are able to bind to the enzyme reversibly and independently as evident by the formation of product quinonoid and external aldimine complexes. We propose that, although D-KAPA binds slower to the enzyme compared with L-KAPA, once bound, it forms a non-productive/catalytically inactive complex with the enzyme such that transimination of this complex, release of the product, and regeneration of the internal aldimine do not occur. The reduction in the regeneration of the internal aldimine would result in lowered affinity of the enzyme for L-alanine and consequently reduced catalysis.

In contrast to the broad substrate stereospecificity exhibited by the M. tuberculosis KAPA synthase, the subsequent enzyme of the pathway, DAPA synthase, which converts KAPA to DAPA, exhibits high stereospecificity in terms of utilizing the S-stereoisomer of KAPA (L-KAPA) over the R-stereoisomer (D-KAPA) as a substrate but is, however, also subject to stereospecific inhibition by the R-stereoisomer (D-KAPA). Thus our study suggests a novel strategy to simultaneously inhibit multiple steps in the biotin biosynthesis pathway, which in combination would effectively provide greater inhibition efficiency. Development of novel analogues of D-KAPA with greater inhibitory potency could serve as putative therapeutic agents for curbing the growth of dreaded pathogens like M. tuberculosis.


   FOOTNOTES
 
* This work was supported by a grant from the Department of Biotechnology, Government of India (to A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental text and Scheme S1. Back

1 These authors contributed equally to the work. Back

2 To whom correspondence should be addressed. Tel.: 91-11-2671-7102; Fax: 91-11-2617-7626; E-mail: surolia{at}nii.res.in.

3 The abbreviations used are: KAPA, 7-keto-8-aminopelargonic acid; DAPA, 7,8-diaminopelargonic acid; PLP, pyridoxal 5'-phosphate; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); ESI-MS, electrospray ionization mass spectrometry; ITC, isothermal titration calorimetry. Back


   ACKNOWLEDGMENTS
 
We thank the Proteomics facility, Molecular Biophysics Unit, Indian Institute of Science, and Subramanya Prakash for the mass spectra.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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