His-bind Ni2+-NTA resin was purchased from Invitrogen while a QuikChange site-directed mutagenesis kit was obtained from Stratagene. Biotinylated thrombin and streptavidin agarose were from Novagen. Triclosan was a gift from Ciba. trans-2-Dodecenoic acid was purchased from TCI. All other chemical reagents were obtained from Sigma-Aldrich.
Synthesis of trans-2-Dodecenoyl-CoA (DD-CoA) and Lauryl-CoA
DD-CoA and lauryl-CoA were prepared from trans
-2-dodecenoic acid and lauric acid, respectively using the mixed anhydride method (21
). Product formation was confirmed by ESI mass spectrometry.
Cloning, Expression and Purification of bmFabV
The entire putative fabV gene from Burkholderia mallei ATCC 23344 (NCBI Reference Sequence: YP_102617.1) was amplified using the primers listed in and inserted into Novagen pET15b vector using the 5’ NdeI and 3’ BamHI restriction sites (underlined) so that a His-tag was encoded at the N-terminus of the coding sequence. After purification from XL1Blue cells (Stratagene) using a DNA purification and gel extraction kit (Qiagen Inc) the correct sequence of the insert was confirmed using ABI DNA sequencing.
Primers Used for Cloning and Mutagenesis
Protein expression was performed using E. coli BL21(DE3)pLysS cells. After transformation, a single colony was used to inoculate 10 ml of Luria Broth (LB) media containing 0.2 mg/ml ampicillin in a 50 ml falcon tube, which was then incubated overnight at 37°C in a floor shaker. The overnight culture was then used to inoculate 1 l of LB media containing ampicillin (0.2 mg/ml) which was incubated on an orbital shaker 37°C until the optical density at 600 nm (OD600) increased to around 1.0. Protein expression was induced by adding 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) and the culture was then shaken at 25°C for 16 h. Cells were harvested by centrifugation at 5,000 rpm for 25 min at 4 ºC. The cell paste was then resuspended in 30 ml of His-binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris HCl, pH 7.9) and lysed by sonication. Cell debris was removed by centrifugation at 33,000 rpm for 60 min at 4 ºC. The resulting supernatant was loaded onto a His-bind column (1.5 cm × 15 cm) containing 4 ml of His-bind resin (Novagen) that had been charged with 9 ml of charge buffer (Ni2+). The column was washed with 60 ml of His-binding buffer and 30 ml of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris HCl, pH 7.9). Subsequently, the protein was eluted using a gradient of 20 ml binding buffer and 40 ml elute buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris HCl, pH 7.9). Fractions containing bmFabV were collected and imidazole was removed using a Sephadex G-25 column (1.5 cm × 55 cm long) using PIPES buffer (30 mM PIPES, 150 mM NaCl, 1.0 mM EDTA pH 8.0) as the eluent. The purity of the protein was shown to be >95% by 12% SDS-PAGE, which gave an apparent molecular weight of ~45 kDa. The concentration of the protein was determined by measuring the A280 and using an extinction coefficient (ε280) of 42,650 M−1cm−1 calculated from the primary sequence. The enzyme was stored at −80°C after flash freezing with liquid N2.
To remove the N-terminal His-tag, 1 µl of biotinylated thrombin was added to 1 ml of bmFabV (20 µM) in PIPES buffer and the reaction mixture was incubated at room temperature for 16 h. Streptavidin agarose (20 µl) was then added and the solution was incubated for an additional hour, after which the agarose was removed by centrifugation at 13,000 rpm for 10 min. The supernatant was further purified by chromatography on a Sephadex G-25 column (0.5 cm × 15 cm long) using PIPES buffer as the eluent, and fractions containing bmFabV lacking the His-tag were pooled and analyzed by 12% SDS-PAGE.
Site-Directed Mutagenesis, Expression and Purification of bmFabV Mutants
The bmFabV mutants Y235A, Y235S, K244A, K244R, K245M and K244A/K245A were prepared using the QuikChange mutagenesis kit from Stratagene with the primers listed in . The sequence of each mutant plasmid was confirmed by ABI DNA sequencing and the expression and purification of all the mutants followed the same procedure as that described above for the wild-type enzyme.
Cloning, Expression and Purification of ftuACP
The open reading frame (NCBI Reference Sequence: YP_170325) which encodes the putative acyl carrier protein (ACP) in Francisella tularensis SCHU S4 was amplified using the primers listed in . The protein product of this ORF shows 68% identity and 79% similarity to the ACP from Burkholderia mallei ATCC 23344. The purified PCR product was inserted into the Novagen pET23b vector using the 5’ BamHI and 3’ XhoI restriction sites (underlined) so that with a His-tag was encoded at the C-terminus of the protein. The Sequence of the construct was confirmed by ABI DNA sequencing.
Expression and purification of ftuACP followed a similar protocol to that described above for bmFabV except that 0.1 M potassium phosphate buffer, pH of 8.0 was used for G-25 chromatography. The purified protein was analyzed by 15% SDS-PAGE and MALDI-TOF mass spectrometry. The concentration of the protein was determined by measuring the absorption at 280 nm and by using an extinction coefficient of 2,560 M−1cm−1 calculated from the primary sequence. The protein was stored at 4°C for at over 3 months without losing any activity.
Preparation of Crotonyl-ACP (Cr-ACP)
Purified ftuACP was concentrated to 900 µM in the reaction buffer (0.1 M potassium phosphate solution at pH of 8.0, 3 ml) and an equimolar amount of dithiothreitol was added to the solution. The reaction mixture was then stirred under nitrogen at 0°C for 2 h to ensure complete reduction of the ACP thiol group, after which a 1.5-fold molar excess of crotonic anhydride was added to the reaction mixture. After stirring for 15 min at 0°C, small molecules were removed from the Cr-ACP by chromatography on a Sephadex G-25 column (0.5 cm × 15 cm) using PIPES buffer as the eluent..
Steady-state Kinetic Analysis
Steady-state kinetic parameters were determined at 25°C in 30 mM PIPES buffer pH 7.9 containing 1.0 mM EDTA and 0.62 mM NaCl. The optimum value for ionic strength was determined by varying the concentration of NaCl from 0 to 700 mM while the pH optimum was obtained by varying the pH of the reaction mixture from 6.6 to 8.5. Initial velocities were determined using a Cary 300 Bio (Varian) spectrophotometer to monitor the oxidation of NADH to NAD+ at 340 nm (ε = 6300 M−1cm−1). Initial characterization of the enzyme mechanism was performed in reaction mixtures containing 5 nM bmFabV and measuring initial velocities at several fixed concentrations of NADH (33, 110 and 250 µM) and by varying the concentration of DD-CoA (1.5–35 µM), or at a fixed concentration of DD-CoA (6, 12 and 18 µM) and by varying the concentration of NADH (10–352 µM). Double reciprocal plots were then used to differentiate between Ping-Pong or ternary-complex mechanisms.
To further investigate the binding order of substrates, product inhibition studies were performed in which each substrate concentration was varied in the presence of several fixed concentrations of one of the products, NAD+ (0, 50 and 110 µM) or lauryl-CoA (0, 50 and 100 µM). The type of inhibition in each case was subsequently determined using a Lineweaver-Burk plot.
Finally the kinetic data in the absence of products were globally fit to the equation for the steady-state sequential Bi Bi mechanism (equation 1)
to determine the Km
values for DD-CoA and NADH.
In equation 1
, v is the initial velocity, Vmax
is the maximum velocity, [A] and [B] are the concentration of the two substrates, KA
are the Michaelis constants for A and B respectively, and KiA
is the dissociation constant for A. Data analysis was performed using GraFit 4.0 (Erithacus)
values of wild-type bmFabV toward DD-CoA and NADH were also determined by varying the concentration of one substrate at a fixed, saturating, concentration (> 10 × Km
) of the second substrate. Data sets were then fit to the Michaelis-Menten equation (equation 2)
using GraFit (Erithacus).
Km values determined using the above method were found to be very close to those obtained from global fitting. Consequently, all subsequent measurements of Km utilized initial velocities determined at fixed saturating concentrations of one substrate while the concentration of the other was varied.
Circular Dichroism Spectroscopy
The far-UV CD spectra of the wild-type bmFabV protein and its mutants were recorded in 50 mM Tris buffer pH 7.9 at 25°C at a concentration of 10 µM using an AVIV 62 DS spectrometer equipped with a Peltier temperature control unit. Data analysis was performed using Microsoft Excel.
Fluorescence Titration of Triclosan Binding to wild-type bmFabV
Equilibrium fluorescence titrations were conducted using a Spex FL3–21 Fluorolog-3 spectrofluorimeter. One µl aliquots of triclosan (20.0 mM stock in DMSO) were added to a 1 ml solution of enzyme (2 µM) in the same buffer as that used in the steady-state kinetic experiments. The excitation wavelength was 295 nm (5 nm slit width), and the emission wavelength was fixed at 335 nm (1 nm slit width). Dilution of protein concentration was controlled to minimum (< 1%) and the change in fluorescence as a function of triclosan concentration was fit to a quadratic equation (equation 3)
In equation 3
are the fluorescent intensity in the presence and absence of enzyme respectively, Fe,max
are the maximum fluorescence intensity in the presence and absence of enzyme, respectively, Kd
is the dissociation constant, [E]0
is the total enzyme concentration and [L] is the amount of triclosan added to the reaction buffer. Data fitting was performed using Grafit.
Progress curve analysis
Progress curve analysis was used to determine if triclosan was a slow onset inhibitor of bmFabV. FabI from F. tularensis (ftuFabI) was used as a model system since it has been shown that triclosan is a slow onset inhibitor of ftuFabI. In the assay, a very low concentration of enzyme (2 nM) and high concentration of substrate (200 µM of DD-CoA and 250 µM of NADH) were used so that initial velocities were linear over a period of 1 h. Since triclosan and other diphenyl ether inhibitors bind to enoyl-ACP reductases in the presence of the oxidized cofactor, 100 µM of NAD+ was added to the reaction so that [NAD+] was effectively constant during progress curve data collection. The concentration of triclosan was 60 µM, and data were collected for 1 h to ensure that the system had reached the steady state. Grafit was used for the data fitting.
Inhibition of bmFabV by Triclosan
Steady state kinetics revealed that triclosan is an uncompetitive inhibitor of bmFabV with respect to DD-CoA and a competitive inhibitor with respect to NADH. Initial velocities were measured at a fixed concentration of NADH (250 µM) or DD-CoA (35 µM) and at various concentrations of the second substrate and inhibitor. The equilibrium constant for the uncompetitive inhibition of bmFabV by triclosan (Kii
) was calculated using equation 4
for the data collected at a fixed concentration of NADH,
where [S] is the concentration of DD-CoA, Km
is the Michaelis-Menten constant for DD-CoA, Vmax
is the maximum velocity, [I] is the concentration of inhibitor added and Kii
is the inhibition constant.
Similarly, the equilibrium constant for the competitive inhibition of bmFabV by triclosan (Kis
) was calculated using equation 5
for the data collected at a fixed concentration of DD-CoA,
where [S] is the concentration of NADH, Km
is the Michaelis-Menten constant for NADH, Vmax
is the maximum velocity, [I] is the concentration of inhibitor added and Kis
is the inhibition constant.