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Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis
Enzyme and Microbial Technology 78 (2015) 18–26
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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis In-Pil Jung a , Jun-Haeng Cho a , Bon-Sung Koo b , Moon-Young Yoon a,∗ a b
Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea Department of Agricultural Biotechnology, National Academy of Agricultural Science, Jeonju 560-500, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 April 2015 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online 12 June 2015 Keywords: Acetohydroxyacid Synthase Site-Directed mutagenesis Herbicide-Binding Site Chlorimuron Ethyl
a b s t r a c t Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold. © 2015 Published by Elsevier Inc.
1. Introduction Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, is still a leading cause of death worldwide. Moreover, the emergence of several multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains of tuberculosis has complicated the current therapy regimen [1]. Due to this situation, several efforts are being made to develop effective anti-tuberculosis agents based on new potential targets [2,3]. Recently, M. tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has gained attention as a promising anti-tuberculosis drug target, since it is essential for bacterial growth [4–6]. Acetohydroxyacid synthase is a ThDP-dependent enzyme (AHAS, EC 4.1.3.18) that catalyzes the first essential step in the biosynthesis of branched-chain amino acids (BCAAs) such as
Abbreviations: TB, tuberculosis; M. tuberculosis, Mycobacterium tuberculosis; AHAS, acetohydroxyacid synthase; BCAA, branched-chain amino acid; FAD, flavin adenine dinucleotide; ThDP, thiamin diphosphate; DMSO, dimethyl sulfoxide. ∗ Corresponding author. Fax: +82 2 2298 0319. E-mail address: [email protected] (M.-Y. Yoon). http://dx.doi.org/10.1016/j.enzmictec.2015.06.009 0141-0229/© 2015 Published by Elsevier Inc.
leucine, isoleucine, and valine. The reactions catalyzed by AHAS include the synthesis of (2S)-acetolactate (AL) from two molecules of pyruvate and the synthesis of (2S)-2-aceto-2-hydroxybutyrate (AHB) from pyruvate and 2-ketobutyrate (KB). AL and AHB are obligatory intermediates in valine and isoleucine biosynthesis, respectively [7–9]. The crystal structures of yeast and Arabidopsis thaliana AHAS have revealed the locations of the active site, including the position and orientation of the cofactors (ThDP, Mg2+ , and FAD) [10–12]. Several studies of ThDP-dependent enzymes have suggested the catalytic mechanism of interaction of ThDP for AHAS [13,14]. Briefly, a highly conserved Q86 residue in the active site plays a key role for catalysis by interacting with the N1 atom of pyrimidine ring in ThDP through the formation of a hydrogen bond [15]. Since AHAS is not expressed in humans, this enzyme has remained as a safe target in use of many commercial herbicides including sulfonylureas, trizolpyrimidines, and imidizolinones for over three decades [16]. Interestingly, many of these herbicides and herbicide-like compounds have also shown inhibitory activity as promising lead compounds for developing novel antibacterial agents [17–20]. Notably, chlorimuron ethyl (CE), a member of the sulfonylurea (SU) family in herbicides, has been widely tested and
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found to be extremely effective with nanomolar range of IC50 against plant, fungal, and bacterial AHASs. Previous studies have identified the key residues involved in CE binding in plant and yeast AHASs [10,11]. However, the interaction of M. tuberculosis AHAS and CE has not yet been characterized, nor have the effects of this herbicide on M. tuberculosis AHAS been determined. Furthermore, many previous studies revealed that herbicide binding site of AHAS was located at dimer interface composed of two catalytic subunits [10–12]. Additionally, our previous work proved that the specific activity of holoenzyme of M. tuberculosis AHAS was not much different from that of catalytic subunit, exhibiting only 1.6 folds increased specific activity. The inhibition properties against herbicides were also similar between holoenzyme and catalytic subunit of M. tuberculosis AHAS [20]. Therefore, we selected several residues (A146, Q148, R318, M512, and V513) in catalytic subunit of M. tuberculosis AHAS conserved with herbicide binding site in A. thaliana AHAS [11] and performed site-directed mutagenesis to determine their effects on the catalytic activity and herbicide binding to M. tuberculosis AHAS.
2. Materials and methods 2.1. Reagents Taq polymerase and Pfu polymerase used for PCR were obtained from Bioneer (Korea) and restriction endonucleases were purchased from Takara (Japan). Gene-specific primers were designed by the Macrogen (Korea), and Escherichia coli BL21 (DE3) and DH5␣ cells were purchased from Invitrogen Co. (USA). Bacto-tryptone, bacto-agar, and yeast extract were obtained from Difco Laboratories (USA), and the most of chemical reagents, sodium pyruvate, flavin adenine dinucleotide (FAD), thiamine diphosphate (ThDP), magnesium chloride, creatine, ␣-naphthol, and potassium phosphate were purchased from Sigma (USA) All other chemicals were analytical grade.
2.2. Homology modeling and multiple sequence alignment To identify a suitable template and construct a homology model, BLAST alignment of AHAS sequences from various species was performed using the Bio Edit program. Homology models of wild type and mutant enzymes were constructed using the Swiss PDB online server [21] with the yeast AHAS (PDB ID: 1T9B) crystal structure as a template. M. tuberculosis AHAS sequence was aligned with the AHAS sequences from various other species using Clustal W2 (Bio Edit Program). The GenBank IDs of AHAS sequences were as follows: A. thaliana (GI: 124372), S. cerevisiae (GI: 296147225), E. coli (GI: 13364177), M. avium (GI: 494536823), and M. tuberculosis (GI: 61226663).
2.3. Site-directed mutagenesis A pET28␣ vector containing M. tuberculosis AHAS cDNA was used as the template for PCR-based site-directed mutagenesis. 100 ng of template DNA, 0.8 M of forward and reverse primers, and 0.8 mM of dNTP were mixed in 25 L reaction volumes. Thermocycling parameters consisted of 20 cycles of the following: 95 ◦ C for 30 s, 58 ◦ C for 1 min, and 72 ◦ C for 10 min. The PCR products were transformed into DH5␣ competent cells and DNA was extracted by the alkaline lysis method. DNA sequencing was carried out by the dideoxy chain-termination procedure at Macrogen.
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2.4. Expression and purification of the catalytic subunit of M. tuberculosis AHAS E. coli BL21 (DE3) cells transformed with a plasmid driving the expression of the catalytic subunit of M. tuberculosis AHAS were grown in Luria-Bertani (LB) medium containing kanamycin (50 g mL−1 ) at 37 ◦ C until the OD600 reached 0.7 – 0.8. To induce protein expression, 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) was added into the medium and the cells were grown at 18 ◦ C overnight. Cells were then harvested by centrifugation at 4000 rpm for 20 min and washed with PBS. The harvested cells were stored at −80 ◦ C. Cell lysis and purification of recombinant proteins were performed as described previously [20].
2.5. Enzymatic assay and characterization of kinetic parameters The activities of wild type M. tuberculosis AHAS and mutant versions were measured by a discontinuous colorimetric assay as described previously [20]. Briefly, each enzyme and the reaction buffer (100 mM potassium phosphate (pH 7.4), 100 mM pyruvate, 5 mM MgCl2 , 1 mM ThDP, and 50 M FAD) were incubated separately at 37 ◦ C for 10 min. The reaction was initiated by adding the enzyme (0.5 g) to the reaction mixture and incubation at 37 ◦ C for 1 h. The final reaction volume was 200 L. The reaction was terminated by addition of 600 mM H2 SO4 , and then further incubated at 65 ◦ C for 15 min, thereby producing acetoin. Next, 100 L of the reaction product was mixed with 90 L of 0.5% (w/v) creatine and 90 L of 5% (w/v) ␣-napthol (in 2.5 M NaOH) and incubated at 65 ◦ C for 15 min. The amount of the acetoin product (525nm = 20,000 M−1 cm−1 ) was measured at 525 nm using a Spectramax M2 spectrophotometer (Molecular Devices, USA).
2.6. Herbicide inhibition kinetics against the mutant versions of AHAS The effects of sulfonylurea herbicides on each variant enzyme of AHAS were tested by adding different concentrations of the herbicides. The selected chemical was dissolved in dimethyl sulfoxide (DMSO). The effect of each herbicide on AHAS activity was determined by the method described above. The 50% inhibition concentration (IC50 ) of each herbicide against M. tuberculosis AHAS was determined by fitting the data to Eq. (1), where V0 is the reaction rate without inhibitor, Vf is the rate at maximal inhibition, and [I] is the inhibitor concentration. V=
(V0 − Vf ) × IC50 + Vf IC50 + [1]
(1)
2.7. Molecular docking Molecular docking was performed using Auto Dock Vina with combined optimization algorithm, including genetic algorithm and simulated annealing [22]. The flexible and nonflexible pocket site residues were identified and their appropriate charges were added prior to herbicide docking. The ligands were modeled with all torsional bonds free in the structure. The docking area was calculated with a three-dimensional grid box with grid points of 120 × 120 × 120 Å and a line spacing of 0.375 Å. Additionally, calculations were grouped on the basis of root mean square deviation (RMSD) over 50 runs. The docking results were analyzed based on the binding energy and the number of hydrogen bond and – interaction.
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Fig. 1. Homology model of M. tuberculosis AHAS (yellow) aligned with the crystal structure of S. cerevisiae AHAS (cyan). Cofactors: ThDP (purple), Mg2+ (green sphere near ThDP), and FAD (green). Herbicide: chlorimuron ethyl (blue). The blue inset shows the herbicide-binding site, including the residues selected for mutation.
3. Results 3.1. Multiple sequence alignment and homology modeling Sequence analysis showed that yeast AHAS had the highest sequence identity (42%) with M. tuberculosis AHAS. Hence, homology modeling of M. tuberculosis AHAS was performed based on the crystal structure of yeast AHAS (PDB ID: 1T9B) as a template. The homology models of each enzyme were evaluated by calculating root mean square deviation (RMSD). All the homology models exhibited RMSD values below 1 Å. The homologous M. tuberculosis AHAS structure showed a similar overall topology to that of the yeast AHAS crystal structure (Fig. 1). The structural validity was analyzed by ramanchandran plot. All the homology model showed residues above 98% in allowed region (Figs. S1–S4). The sequence alignment also indicated that the targeted binding site residues
for herbicides were highly conserved across AHASs from various species (Fig. 2). This result suggested that the selected residues (A146, Q148, R318, M512, and V513) might play a crucial role in M. tuberculosis AHAS. 3.2. Mutagenesis and purification of mutant versions of M. tuberculosis AHAS To evaluate the functional roles of A146, Q148, R318, M512, and V513, these residues were replaced by various similar and oppositely charged residues using site-directed mutagenesis. Specifically, A146 was substituted with V146 and E146; Q148 was replaced by A148, E148, and N148; R318 was substituted to A318, H318, and E318; and M512 and V513 were changed to L512 and L513, respectively. Each of mutants was expressed in soluble form and purified using Ni+ -chelating column chromatography.
Fig. 2. Sequence alignment of M. tuberculosis AHAS with various AHAS. A. thaliana AHAS (plant), S. cerevisiae AHAS (yeast), E. coli AHAS, and M. avium AHAS (Bacteria). Red boxes indicate conserved residues that were selected for mutation.
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Fig. 3. SDS-PAGE analysis of the different purification stages of M. tuberculosis AHAS point mutants. Electrophoresis was performed using a 12% polyacrylamide gel and proteins were stained with Coomassie blue R-250. Lane M, protein ladder; lane 1, M. tuberculosis wild type AHAS; lanes 2 – 4, R318A, R318H, and R318E; lanes 5 and 6, A146 V and A146E; lanes 7 – 9, Q148A, Q148N, and Q148E; lanes 10 and 11, M512L and V513L, respectively.
Table 1 Kinetic parameters of wild type and mutant versions of M. tuberculosis AHAS.
Wild type A146V A146E Q148A Q148N Q148E M512L V513L
Specific activity (U × mg−1 )
Km (mM)
Ks for ThDP (M)
Ks for Mg2+ (mM)
IC50 for CE (M)
IC50 for IQ (mM)
2.8 0.38 0.11 0.76 0.14 0.18 0.75 0.70
2.76 20.5 22.0 23.4 21.0 31.0 9.09 11.7
51.23 61.89 68.14 59.27 85.54 61.03 134.3 167.2
0.27 0.16 0.31 0.25 0.33 0.20 1.13 1.09
8.97 70.6 161 130 73.1 N.D 40.7 17.9
0.23 3.03 N.D
N.D, not determined.
The molecular weight of each mutant was determined to be 68 kDa, which is similar to that of the catalytic subunit of wild type AHAS, by SDS-PAGE (Fig. 3). 3.3. Determination of kinetic parameters of the mutant AHASs Each AHAS mutant was functionally assessed by analyzing its kinetic parameters towards the appropriate substrate and cofactor. Remarkably, none of the R318 point mutants (R318A, R318H,
Fig. 4. Saturation curves as a function of pyruvate concentration for the M. tuberculosis AHAS point mutants. The solid lines show the best fits of the Michaelis–Menten equation. (a) Km values = 20.5 mM for A146 V, 22.0 mM for A146E, 23.4 mM for Q148A, 21.0 mM for Q148N, 31.0 mM for Q148E, 9.09 mM for M512L, and 11.7 mM for V513L.
and R318E) exhibited any activity. This result indicates that R318 is essential for the catalytic activity of AHAS. All the other point mutants showed lower specific activities than wild type AHAS. In particular, the A146E, Q148N, and Q148E mutants showed a significant decrease (20-fold) in specific activity (Table 1). Analysis of the substrate kinetics of the A146 and Q148 mutants revealed that these variants exhibited decreased affinity towards the substrate (pyruvate), with maximum 11-fold increased Km values compared with the wild type. Similarly, the M512L and V513L mutants also showed slightly decreased affinity towards the substrate, with 3-
Fig. 5. Saturation curves as a function of ThDP concentration for the M. tuberculosis AHAS point mutants. Ks for ThDP = 61.9 M for A146 V, 68.1 M for A146E, 59.3 M for Q148A, 85.5 M for Q148N, 61.3 M for Q148E, 134 M for M512L, and 150 M for V513L.
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Fig. 6. Saturation curves as a function of Mg2+ concentration for the M. tuberculosis AHAS point mutants. Ks for Mg2+ = 0.16 mM for A146 V, 0.31 mM for A146E, 0.25 mM for Q148A, 0.33 mM for Q148N, 0.20 mM for Q148E, 1.13 mM for M512L, and 1.09 mM for V513L.
fold higher Km values compared with the wild type (Fig. 4). The effects of cofactors (ThDP and Mg2+ ) on each mutant enzyme were also examined (Figs. 5 and 6). Interestingly, the Ks values for ThDP and for Mg2+ of the A146 and Q148 mutants were hardly affected; however, the M512 and V513 mutants showed 3-fold higher Ks values for ThDP and maximum 4-fold increased Ks values for Mg2+ . Briefly, this result suggests that the A146, Q148, and R318 residues might play a key role in substrate binding, whereas the M512 and V513 residues mediate interaction of cofactors (ThDP and Mg2+ ) with M. tuberculosis AHAS.
3.4. Herbicide inhibition kinetics of the AHAS mutants To analyze the roles of the selected residues, the ability of Chlorimuron Ethyl (CE) and Imazaquin (IQ) to inhibit each mutant version of AHAS was examined. To this end, the IC50 value of CE against each mutant was determined (Table 1). With the exception of the R318 mutant, the IC50 for CE against each mutant was different compared with the value of wild type AHAS. The M512L and V513L mutants exhibited slightly higher IC50 values (2-fold and 4-fold increased, respectively) than wild type AHAS. However,
Fig. 7. Relative activity of the AHAS large subunit (0.5 g) as a function of herbicide (chlorimuron ethyl, CE) concentration. (a) IC50 values = 70.6 M for A146 V, 161 M for A146E, 130 M for Q148A and 73.1 M for Q148N; (b) 40.7 M for M512L and 17.9 M for V513L.
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Fig. 8. Interaction of an herbicide (CE) with the M. tuberculosis AHAS A146 point mutants. (a) wild type AHAS, (b) A146 V, (c) A146E. The binding energy of the herbicide to each mutant version is shown below each panel.
Fig. 9. Interaction of an herbicide (CE) with the M. tuberculosis AHAS Q148 point mutants. (a) wild type AHAS, (b) Q148A, (c) Q148N, (d) Q148E. The binding energy of the herbicide to each mutant version is shown below each panel.
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Fig. 10. Interaction of an herbicide (CE) with the M. tuberculosis AHAS M512 and V513 point mutants. (a) wild type AHAS, (b) M512L, (c) V513L. The binding energy of the herbicide to each mutant version is shown below each panel.
the IC50 values for both the A146 and Q148 mutants were significantly different from the wild type value. Specifically, the A146 V and A146E mutants showed 8-fold and 18-fold higher IC50 values compared with wild type AHAS, respectively. Similarly, the Q148A and Q148N mutants exhibited 14-fold and 8-fold increased IC50 values, respectively. Surprisingly, the Q148E mutant was not inhibited by CE (Fig. 7). Additionally, all the variant enzymes exhibited significantly increased resistance against IQ. Although wild type showed IC50 of 0.23 mM, only A146 V exhibited inhibition of IQ with 14fold increased IC50 and the rest of them did not show inhibition at all. This result indicates that A146 and Q148 mediate the interaction with herbicides via hydrophilic interactions and hydrophobic interactions, respectively.
3.5. Molecular docking The effects of each point mutation on herbicide binding were examined by molecular docking. Based on binding energy and the number of hydrogen bond and – interaction, the best binding mode of CE was selected among approximately 105 random seed. As shown in Figs. 8 and 9, the binding mode of CE was slightly altered for the A146 and Q148 mutants. In particular, the oppositely charged mutants (A146E and Q148E) showed the highest binding energies (−6.1 kcal mol−1 and −5.7 kcal mol−1 , respectively), indicating that the herbicide binds with less affinity to these mutants. However, CE binding to the M512 and V513 mutants was hardly affected; these mutants exhibited similar binding energies to the wild type version (M512L, −8.4 kcal mol−1 ; V513L, −8.5 kcal mol−1 ; wild type, −8.5 kcal mol−1 ) (Fig. 10). Both the binding mode and
the binding energy were significantly affected for the R318 mutant (Fig. 11). Moreover, the R318 mutation also resulted in a slight conformational change in the substrate-accessing channel (Fig. 12). As a result, the distance between ThDP and the mutated residue in the R318A, R318H, and R318E mutants was larger than wild type AHAS. Taken together, these results suggest that the R318 residue may mediate the herbicide interaction and may also play a role in the catalytic activity of AHAS.
4. Discussion Previous crystallographic studies of A. thaliana and yeast AHAS [10–12] have revealed important residues involved in herbicide binding. However, the crystal structure of M. tuberculosis AHAS has not yet been solved. Recent studies have suggested that many herbicide and herbicide-like compounds exert significant inhibitory action against M. tuberculosis AHAS and also against various other TB strains [4]; however, the exact mechanisms of interaction of these herbicides and inhibitors with M. tuberculosis AHAS are not yet fully understood. Furthermore, previous studies have also reported that some of these herbicide-binding residues are involved in the development of herbicide resistance [23]. Thus, in this study, we examined the roles of conserved residues in the herbicide-binding site of M. tuberculosis AHAS by site-directed mutagenesis. All mutant enzymes were purified to homogeneity, and the molecular weight of each mutant was similar to that of wild type AHAS (68 kDa). The R318 residue is a well-known residue that is conserved in the AHASs of various species and is known to mediate inter-
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Fig. 11. Interaction of an herbicide (CE) with the M. tuberculosis AHAS R318 point mutants. (a) wild type AHAS, (b) R318A, (c) R318H, (d) R318E. The binding energy of the herbicide to each mutant version is shown below each panel.
action of herbicides with AHAS [11]. This residue has also been identified to play an important catalytic role in plant AHAS activity. Importantly, substitution of this residue by various similar or oppositely charged residues resulted in a complete loss of the enzymatic activity of M. tuberculosis AHAS. Furthermore, molecular docking analysis revealed that substitution of this residue not only affected the binding mode of an herbicide (CE) but also influenced the distance between this residue and the active site in M. tuberculosis AHAS. In agreement with this finding, one previous study suggested that the homologous residue in E. coli AHAS (R276) interacts with the carboxylate side chain of the second substrate of AHAS [13]. Based on these results, we hypothesize that M. tuberculosis AHAS R318 may be involved in the catalytic activity of this enzyme. Among the residues studied here, A146 and Q148 were also conserved in various AHASs and are known to be key residues for herbicide binding via hydrophobic interactions [11]. All point mutations of these residues resulted in lower specific activity and higher Km values (Table 1). Furthermore, the IC50 values for CE values were significantly increased by mutation of these residues, indicating that these residues mediate herbicide interactions with M. tuberculosis AHAS. Strikingly, the Q148E point mutant was not inhibited by CE. Also, docking studies of these mutants revealed that Q148 was positioned close to the herbicide; moreover, these mutant enzymes showed higher binding energies than wild type AHAS. The two A146 point mutants tested exhibited higher binding energies than the wild type enzyme (Fig. 8). Moreover, the binding energy of A146E was higher than that of A146 V, which corresponded with the IC50 data. These results indicate that the hydrophobic properties of the alanine residue could
be important for the interaction between M. tuberculosis AHAS and CE. Finally, we also evaluated the roles of the M512 and V513 residues in M. tuberculosis AHAS. Interestingly, these residues also exhibited conserved interactions with herbicides in plant AHAS [16]. However, surprisingly, substitution of these residues did not greatly affect the inhibition kinetics. Specifically, the M512L and V513L point mutants showed only 4-fold and 2-fold higher IC50 values than the wild type, respectively. Also, docking studies revealed that the binding energies of point mutants were not significantly affected. Therefore, we conclude that the M512 and V513 residues constitute part of the herbicide-binding site in M. tuberculosis AHAS, but they do not interact directly with the herbicide. In conclusion, mutational analysis of multiple known herbicidebinding residues led to altered kinetic parameters and inhibition properties of M. tuberculosis AHAS by a well-known herbicide, CE. The altered kinetic parameters for various point mutants towards substrate and cofactors indicated that the selected residues might have some catalytic function in M. tuberculosis AHAS. R318 appears to be a particularly important residue, since point mutations of this residue resulted in a complete loss of enzymatic activity. In addition, the point mutants tested demonstrated significant resistance against CE compared with wild type AHAS, indicating that these residues might be involved in herbicide binding. Collectively, the results presented here suggest that the residues examined might play an important role in CE binding to AHAS and are also likely to be involved in the catalytic activity of AHAS.
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Fig. 12. Conformations of the R318 point mutants. The yellow CPK model indicates the following residues: (a) R318, (b) A318, (c) H318, and (d) E318. The stick model indicates ThDP.
Acknowledgement This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01133002)” funded by the Rural Development Administration. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec.2015. 06.009 References [1] P.J. Dolin, M.C. Raviglione, A. Kochi, Global tuberculosis incidence and mortality during 1990–2000, Bull. World Health Organ. 72 (1994) 213–220. [2] D.H. Kim, H.J. Kim, S.K. Park, S.J. Kong, Y.S. Kim, T.H. Kim, E.K. Kim, K.M. Lee, S.S. Lee, J.S. Park, W.J. Koh, C.H. Lee, J.Y. Kim, T.S. Shim, Treatment outcomes and long-term survival in patients with extensively drug-resistant tuberculosis, Am. J. Respir. Crit. Care Med. 178 (2008) 1075–1082. [3] A. Zumla, P. Nahid, S.T. Cole, Advances in the development of new tuberculosis drugs and treatment regimens, Nat. Rev. Drug Discov. 12 (2013) 388–404. [4] N. Pue, L.W. Guddat, Acetohydroxyacid synthase: a target for antimicrobial drug discovery, Curr. Pharm. Des. (2014) 740–753. [5] F.C. Bange, A.M. Brown, W.R. Jacobs Jr, Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages, Infect. Immun. 64 (1996) 1794–1799. [6] I. Guleria, R. Teitelbaum, R.A. McAdam, G. Kalpana, W.R. Jacobs Jr, B.R. Bloom, Auxotrophic vaccines for tuberculosis, Nat. Med. 2 (1996) 334–337. [7] D. Chipman, Z. Barak, J.V. Schloss, Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases, Biochim. Biophys. Acta 1385 (1998) 401–419. [8] R.G. Duggleby, S.S. Pang, Acetohydroxyacid synthase, J. Biochem. Mol. Biol. 33 (2000) 1–36. [9] J.A. McCourt, R.G. Duggleby, Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids, Amino Acids 31 (2006) 173–210. [10] S.S. Pang, R.G. Duggleby, L.W. Guddat, Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors, J. Mol. Biol. 317 (2002) 249–262.
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Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis In-Pil Jung a , Jun-Haeng Cho a , Bon-Sung Koo b , Moon-Young Yoon a,∗ a b
Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea Department of Agricultural Biotechnology, National Academy of Agricultural Science, Jeonju 560-500, Republic of Korea
a r t i c l e
i n f o
Article history: Received 7 April 2015 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online 12 June 2015 Keywords: Acetohydroxyacid Synthase Site-Directed mutagenesis Herbicide-Binding Site Chlorimuron Ethyl
a b s t r a c t Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold. © 2015 Published by Elsevier Inc.
1. Introduction Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, is still a leading cause of death worldwide. Moreover, the emergence of several multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains of tuberculosis has complicated the current therapy regimen [1]. Due to this situation, several efforts are being made to develop effective anti-tuberculosis agents based on new potential targets [2,3]. Recently, M. tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has gained attention as a promising anti-tuberculosis drug target, since it is essential for bacterial growth [4–6]. Acetohydroxyacid synthase is a ThDP-dependent enzyme (AHAS, EC 4.1.3.18) that catalyzes the first essential step in the biosynthesis of branched-chain amino acids (BCAAs) such as
Abbreviations: TB, tuberculosis; M. tuberculosis, Mycobacterium tuberculosis; AHAS, acetohydroxyacid synthase; BCAA, branched-chain amino acid; FAD, flavin adenine dinucleotide; ThDP, thiamin diphosphate; DMSO, dimethyl sulfoxide. ∗ Corresponding author. Fax: +82 2 2298 0319. E-mail address: [email protected] (M.-Y. Yoon). http://dx.doi.org/10.1016/j.enzmictec.2015.06.009 0141-0229/© 2015 Published by Elsevier Inc.
leucine, isoleucine, and valine. The reactions catalyzed by AHAS include the synthesis of (2S)-acetolactate (AL) from two molecules of pyruvate and the synthesis of (2S)-2-aceto-2-hydroxybutyrate (AHB) from pyruvate and 2-ketobutyrate (KB). AL and AHB are obligatory intermediates in valine and isoleucine biosynthesis, respectively [7–9]. The crystal structures of yeast and Arabidopsis thaliana AHAS have revealed the locations of the active site, including the position and orientation of the cofactors (ThDP, Mg2+ , and FAD) [10–12]. Several studies of ThDP-dependent enzymes have suggested the catalytic mechanism of interaction of ThDP for AHAS [13,14]. Briefly, a highly conserved Q86 residue in the active site plays a key role for catalysis by interacting with the N1 atom of pyrimidine ring in ThDP through the formation of a hydrogen bond [15]. Since AHAS is not expressed in humans, this enzyme has remained as a safe target in use of many commercial herbicides including sulfonylureas, trizolpyrimidines, and imidizolinones for over three decades [16]. Interestingly, many of these herbicides and herbicide-like compounds have also shown inhibitory activity as promising lead compounds for developing novel antibacterial agents [17–20]. Notably, chlorimuron ethyl (CE), a member of the sulfonylurea (SU) family in herbicides, has been widely tested and
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found to be extremely effective with nanomolar range of IC50 against plant, fungal, and bacterial AHASs. Previous studies have identified the key residues involved in CE binding in plant and yeast AHASs [10,11]. However, the interaction of M. tuberculosis AHAS and CE has not yet been characterized, nor have the effects of this herbicide on M. tuberculosis AHAS been determined. Furthermore, many previous studies revealed that herbicide binding site of AHAS was located at dimer interface composed of two catalytic subunits [10–12]. Additionally, our previous work proved that the specific activity of holoenzyme of M. tuberculosis AHAS was not much different from that of catalytic subunit, exhibiting only 1.6 folds increased specific activity. The inhibition properties against herbicides were also similar between holoenzyme and catalytic subunit of M. tuberculosis AHAS [20]. Therefore, we selected several residues (A146, Q148, R318, M512, and V513) in catalytic subunit of M. tuberculosis AHAS conserved with herbicide binding site in A. thaliana AHAS [11] and performed site-directed mutagenesis to determine their effects on the catalytic activity and herbicide binding to M. tuberculosis AHAS.
2. Materials and methods 2.1. Reagents Taq polymerase and Pfu polymerase used for PCR were obtained from Bioneer (Korea) and restriction endonucleases were purchased from Takara (Japan). Gene-specific primers were designed by the Macrogen (Korea), and Escherichia coli BL21 (DE3) and DH5␣ cells were purchased from Invitrogen Co. (USA). Bacto-tryptone, bacto-agar, and yeast extract were obtained from Difco Laboratories (USA), and the most of chemical reagents, sodium pyruvate, flavin adenine dinucleotide (FAD), thiamine diphosphate (ThDP), magnesium chloride, creatine, ␣-naphthol, and potassium phosphate were purchased from Sigma (USA) All other chemicals were analytical grade.
2.2. Homology modeling and multiple sequence alignment To identify a suitable template and construct a homology model, BLAST alignment of AHAS sequences from various species was performed using the Bio Edit program. Homology models of wild type and mutant enzymes were constructed using the Swiss PDB online server [21] with the yeast AHAS (PDB ID: 1T9B) crystal structure as a template. M. tuberculosis AHAS sequence was aligned with the AHAS sequences from various other species using Clustal W2 (Bio Edit Program). The GenBank IDs of AHAS sequences were as follows: A. thaliana (GI: 124372), S. cerevisiae (GI: 296147225), E. coli (GI: 13364177), M. avium (GI: 494536823), and M. tuberculosis (GI: 61226663).
2.3. Site-directed mutagenesis A pET28␣ vector containing M. tuberculosis AHAS cDNA was used as the template for PCR-based site-directed mutagenesis. 100 ng of template DNA, 0.8 M of forward and reverse primers, and 0.8 mM of dNTP were mixed in 25 L reaction volumes. Thermocycling parameters consisted of 20 cycles of the following: 95 ◦ C for 30 s, 58 ◦ C for 1 min, and 72 ◦ C for 10 min. The PCR products were transformed into DH5␣ competent cells and DNA was extracted by the alkaline lysis method. DNA sequencing was carried out by the dideoxy chain-termination procedure at Macrogen.
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2.4. Expression and purification of the catalytic subunit of M. tuberculosis AHAS E. coli BL21 (DE3) cells transformed with a plasmid driving the expression of the catalytic subunit of M. tuberculosis AHAS were grown in Luria-Bertani (LB) medium containing kanamycin (50 g mL−1 ) at 37 ◦ C until the OD600 reached 0.7 – 0.8. To induce protein expression, 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) was added into the medium and the cells were grown at 18 ◦ C overnight. Cells were then harvested by centrifugation at 4000 rpm for 20 min and washed with PBS. The harvested cells were stored at −80 ◦ C. Cell lysis and purification of recombinant proteins were performed as described previously [20].
2.5. Enzymatic assay and characterization of kinetic parameters The activities of wild type M. tuberculosis AHAS and mutant versions were measured by a discontinuous colorimetric assay as described previously [20]. Briefly, each enzyme and the reaction buffer (100 mM potassium phosphate (pH 7.4), 100 mM pyruvate, 5 mM MgCl2 , 1 mM ThDP, and 50 M FAD) were incubated separately at 37 ◦ C for 10 min. The reaction was initiated by adding the enzyme (0.5 g) to the reaction mixture and incubation at 37 ◦ C for 1 h. The final reaction volume was 200 L. The reaction was terminated by addition of 600 mM H2 SO4 , and then further incubated at 65 ◦ C for 15 min, thereby producing acetoin. Next, 100 L of the reaction product was mixed with 90 L of 0.5% (w/v) creatine and 90 L of 5% (w/v) ␣-napthol (in 2.5 M NaOH) and incubated at 65 ◦ C for 15 min. The amount of the acetoin product (525nm = 20,000 M−1 cm−1 ) was measured at 525 nm using a Spectramax M2 spectrophotometer (Molecular Devices, USA).
2.6. Herbicide inhibition kinetics against the mutant versions of AHAS The effects of sulfonylurea herbicides on each variant enzyme of AHAS were tested by adding different concentrations of the herbicides. The selected chemical was dissolved in dimethyl sulfoxide (DMSO). The effect of each herbicide on AHAS activity was determined by the method described above. The 50% inhibition concentration (IC50 ) of each herbicide against M. tuberculosis AHAS was determined by fitting the data to Eq. (1), where V0 is the reaction rate without inhibitor, Vf is the rate at maximal inhibition, and [I] is the inhibitor concentration. V=
(V0 − Vf ) × IC50 + Vf IC50 + [1]
(1)
2.7. Molecular docking Molecular docking was performed using Auto Dock Vina with combined optimization algorithm, including genetic algorithm and simulated annealing [22]. The flexible and nonflexible pocket site residues were identified and their appropriate charges were added prior to herbicide docking. The ligands were modeled with all torsional bonds free in the structure. The docking area was calculated with a three-dimensional grid box with grid points of 120 × 120 × 120 Å and a line spacing of 0.375 Å. Additionally, calculations were grouped on the basis of root mean square deviation (RMSD) over 50 runs. The docking results were analyzed based on the binding energy and the number of hydrogen bond and – interaction.
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Fig. 1. Homology model of M. tuberculosis AHAS (yellow) aligned with the crystal structure of S. cerevisiae AHAS (cyan). Cofactors: ThDP (purple), Mg2+ (green sphere near ThDP), and FAD (green). Herbicide: chlorimuron ethyl (blue). The blue inset shows the herbicide-binding site, including the residues selected for mutation.
3. Results 3.1. Multiple sequence alignment and homology modeling Sequence analysis showed that yeast AHAS had the highest sequence identity (42%) with M. tuberculosis AHAS. Hence, homology modeling of M. tuberculosis AHAS was performed based on the crystal structure of yeast AHAS (PDB ID: 1T9B) as a template. The homology models of each enzyme were evaluated by calculating root mean square deviation (RMSD). All the homology models exhibited RMSD values below 1 Å. The homologous M. tuberculosis AHAS structure showed a similar overall topology to that of the yeast AHAS crystal structure (Fig. 1). The structural validity was analyzed by ramanchandran plot. All the homology model showed residues above 98% in allowed region (Figs. S1–S4). The sequence alignment also indicated that the targeted binding site residues
for herbicides were highly conserved across AHASs from various species (Fig. 2). This result suggested that the selected residues (A146, Q148, R318, M512, and V513) might play a crucial role in M. tuberculosis AHAS. 3.2. Mutagenesis and purification of mutant versions of M. tuberculosis AHAS To evaluate the functional roles of A146, Q148, R318, M512, and V513, these residues were replaced by various similar and oppositely charged residues using site-directed mutagenesis. Specifically, A146 was substituted with V146 and E146; Q148 was replaced by A148, E148, and N148; R318 was substituted to A318, H318, and E318; and M512 and V513 were changed to L512 and L513, respectively. Each of mutants was expressed in soluble form and purified using Ni+ -chelating column chromatography.
Fig. 2. Sequence alignment of M. tuberculosis AHAS with various AHAS. A. thaliana AHAS (plant), S. cerevisiae AHAS (yeast), E. coli AHAS, and M. avium AHAS (Bacteria). Red boxes indicate conserved residues that were selected for mutation.
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Fig. 3. SDS-PAGE analysis of the different purification stages of M. tuberculosis AHAS point mutants. Electrophoresis was performed using a 12% polyacrylamide gel and proteins were stained with Coomassie blue R-250. Lane M, protein ladder; lane 1, M. tuberculosis wild type AHAS; lanes 2 – 4, R318A, R318H, and R318E; lanes 5 and 6, A146 V and A146E; lanes 7 – 9, Q148A, Q148N, and Q148E; lanes 10 and 11, M512L and V513L, respectively.
Table 1 Kinetic parameters of wild type and mutant versions of M. tuberculosis AHAS.
Wild type A146V A146E Q148A Q148N Q148E M512L V513L
Specific activity (U × mg−1 )
Km (mM)
Ks for ThDP (M)
Ks for Mg2+ (mM)
IC50 for CE (M)
IC50 for IQ (mM)
2.8 0.38 0.11 0.76 0.14 0.18 0.75 0.70
2.76 20.5 22.0 23.4 21.0 31.0 9.09 11.7
51.23 61.89 68.14 59.27 85.54 61.03 134.3 167.2
0.27 0.16 0.31 0.25 0.33 0.20 1.13 1.09
8.97 70.6 161 130 73.1 N.D 40.7 17.9
0.23 3.03 N.D
N.D, not determined.
The molecular weight of each mutant was determined to be 68 kDa, which is similar to that of the catalytic subunit of wild type AHAS, by SDS-PAGE (Fig. 3). 3.3. Determination of kinetic parameters of the mutant AHASs Each AHAS mutant was functionally assessed by analyzing its kinetic parameters towards the appropriate substrate and cofactor. Remarkably, none of the R318 point mutants (R318A, R318H,
Fig. 4. Saturation curves as a function of pyruvate concentration for the M. tuberculosis AHAS point mutants. The solid lines show the best fits of the Michaelis–Menten equation. (a) Km values = 20.5 mM for A146 V, 22.0 mM for A146E, 23.4 mM for Q148A, 21.0 mM for Q148N, 31.0 mM for Q148E, 9.09 mM for M512L, and 11.7 mM for V513L.
and R318E) exhibited any activity. This result indicates that R318 is essential for the catalytic activity of AHAS. All the other point mutants showed lower specific activities than wild type AHAS. In particular, the A146E, Q148N, and Q148E mutants showed a significant decrease (20-fold) in specific activity (Table 1). Analysis of the substrate kinetics of the A146 and Q148 mutants revealed that these variants exhibited decreased affinity towards the substrate (pyruvate), with maximum 11-fold increased Km values compared with the wild type. Similarly, the M512L and V513L mutants also showed slightly decreased affinity towards the substrate, with 3-
Fig. 5. Saturation curves as a function of ThDP concentration for the M. tuberculosis AHAS point mutants. Ks for ThDP = 61.9 M for A146 V, 68.1 M for A146E, 59.3 M for Q148A, 85.5 M for Q148N, 61.3 M for Q148E, 134 M for M512L, and 150 M for V513L.
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Fig. 6. Saturation curves as a function of Mg2+ concentration for the M. tuberculosis AHAS point mutants. Ks for Mg2+ = 0.16 mM for A146 V, 0.31 mM for A146E, 0.25 mM for Q148A, 0.33 mM for Q148N, 0.20 mM for Q148E, 1.13 mM for M512L, and 1.09 mM for V513L.
fold higher Km values compared with the wild type (Fig. 4). The effects of cofactors (ThDP and Mg2+ ) on each mutant enzyme were also examined (Figs. 5 and 6). Interestingly, the Ks values for ThDP and for Mg2+ of the A146 and Q148 mutants were hardly affected; however, the M512 and V513 mutants showed 3-fold higher Ks values for ThDP and maximum 4-fold increased Ks values for Mg2+ . Briefly, this result suggests that the A146, Q148, and R318 residues might play a key role in substrate binding, whereas the M512 and V513 residues mediate interaction of cofactors (ThDP and Mg2+ ) with M. tuberculosis AHAS.
3.4. Herbicide inhibition kinetics of the AHAS mutants To analyze the roles of the selected residues, the ability of Chlorimuron Ethyl (CE) and Imazaquin (IQ) to inhibit each mutant version of AHAS was examined. To this end, the IC50 value of CE against each mutant was determined (Table 1). With the exception of the R318 mutant, the IC50 for CE against each mutant was different compared with the value of wild type AHAS. The M512L and V513L mutants exhibited slightly higher IC50 values (2-fold and 4-fold increased, respectively) than wild type AHAS. However,
Fig. 7. Relative activity of the AHAS large subunit (0.5 g) as a function of herbicide (chlorimuron ethyl, CE) concentration. (a) IC50 values = 70.6 M for A146 V, 161 M for A146E, 130 M for Q148A and 73.1 M for Q148N; (b) 40.7 M for M512L and 17.9 M for V513L.
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Fig. 8. Interaction of an herbicide (CE) with the M. tuberculosis AHAS A146 point mutants. (a) wild type AHAS, (b) A146 V, (c) A146E. The binding energy of the herbicide to each mutant version is shown below each panel.
Fig. 9. Interaction of an herbicide (CE) with the M. tuberculosis AHAS Q148 point mutants. (a) wild type AHAS, (b) Q148A, (c) Q148N, (d) Q148E. The binding energy of the herbicide to each mutant version is shown below each panel.
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Fig. 10. Interaction of an herbicide (CE) with the M. tuberculosis AHAS M512 and V513 point mutants. (a) wild type AHAS, (b) M512L, (c) V513L. The binding energy of the herbicide to each mutant version is shown below each panel.
the IC50 values for both the A146 and Q148 mutants were significantly different from the wild type value. Specifically, the A146 V and A146E mutants showed 8-fold and 18-fold higher IC50 values compared with wild type AHAS, respectively. Similarly, the Q148A and Q148N mutants exhibited 14-fold and 8-fold increased IC50 values, respectively. Surprisingly, the Q148E mutant was not inhibited by CE (Fig. 7). Additionally, all the variant enzymes exhibited significantly increased resistance against IQ. Although wild type showed IC50 of 0.23 mM, only A146 V exhibited inhibition of IQ with 14fold increased IC50 and the rest of them did not show inhibition at all. This result indicates that A146 and Q148 mediate the interaction with herbicides via hydrophilic interactions and hydrophobic interactions, respectively.
3.5. Molecular docking The effects of each point mutation on herbicide binding were examined by molecular docking. Based on binding energy and the number of hydrogen bond and – interaction, the best binding mode of CE was selected among approximately 105 random seed. As shown in Figs. 8 and 9, the binding mode of CE was slightly altered for the A146 and Q148 mutants. In particular, the oppositely charged mutants (A146E and Q148E) showed the highest binding energies (−6.1 kcal mol−1 and −5.7 kcal mol−1 , respectively), indicating that the herbicide binds with less affinity to these mutants. However, CE binding to the M512 and V513 mutants was hardly affected; these mutants exhibited similar binding energies to the wild type version (M512L, −8.4 kcal mol−1 ; V513L, −8.5 kcal mol−1 ; wild type, −8.5 kcal mol−1 ) (Fig. 10). Both the binding mode and
the binding energy were significantly affected for the R318 mutant (Fig. 11). Moreover, the R318 mutation also resulted in a slight conformational change in the substrate-accessing channel (Fig. 12). As a result, the distance between ThDP and the mutated residue in the R318A, R318H, and R318E mutants was larger than wild type AHAS. Taken together, these results suggest that the R318 residue may mediate the herbicide interaction and may also play a role in the catalytic activity of AHAS.
4. Discussion Previous crystallographic studies of A. thaliana and yeast AHAS [10–12] have revealed important residues involved in herbicide binding. However, the crystal structure of M. tuberculosis AHAS has not yet been solved. Recent studies have suggested that many herbicide and herbicide-like compounds exert significant inhibitory action against M. tuberculosis AHAS and also against various other TB strains [4]; however, the exact mechanisms of interaction of these herbicides and inhibitors with M. tuberculosis AHAS are not yet fully understood. Furthermore, previous studies have also reported that some of these herbicide-binding residues are involved in the development of herbicide resistance [23]. Thus, in this study, we examined the roles of conserved residues in the herbicide-binding site of M. tuberculosis AHAS by site-directed mutagenesis. All mutant enzymes were purified to homogeneity, and the molecular weight of each mutant was similar to that of wild type AHAS (68 kDa). The R318 residue is a well-known residue that is conserved in the AHASs of various species and is known to mediate inter-
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Fig. 11. Interaction of an herbicide (CE) with the M. tuberculosis AHAS R318 point mutants. (a) wild type AHAS, (b) R318A, (c) R318H, (d) R318E. The binding energy of the herbicide to each mutant version is shown below each panel.
action of herbicides with AHAS [11]. This residue has also been identified to play an important catalytic role in plant AHAS activity. Importantly, substitution of this residue by various similar or oppositely charged residues resulted in a complete loss of the enzymatic activity of M. tuberculosis AHAS. Furthermore, molecular docking analysis revealed that substitution of this residue not only affected the binding mode of an herbicide (CE) but also influenced the distance between this residue and the active site in M. tuberculosis AHAS. In agreement with this finding, one previous study suggested that the homologous residue in E. coli AHAS (R276) interacts with the carboxylate side chain of the second substrate of AHAS [13]. Based on these results, we hypothesize that M. tuberculosis AHAS R318 may be involved in the catalytic activity of this enzyme. Among the residues studied here, A146 and Q148 were also conserved in various AHASs and are known to be key residues for herbicide binding via hydrophobic interactions [11]. All point mutations of these residues resulted in lower specific activity and higher Km values (Table 1). Furthermore, the IC50 values for CE values were significantly increased by mutation of these residues, indicating that these residues mediate herbicide interactions with M. tuberculosis AHAS. Strikingly, the Q148E point mutant was not inhibited by CE. Also, docking studies of these mutants revealed that Q148 was positioned close to the herbicide; moreover, these mutant enzymes showed higher binding energies than wild type AHAS. The two A146 point mutants tested exhibited higher binding energies than the wild type enzyme (Fig. 8). Moreover, the binding energy of A146E was higher than that of A146 V, which corresponded with the IC50 data. These results indicate that the hydrophobic properties of the alanine residue could
be important for the interaction between M. tuberculosis AHAS and CE. Finally, we also evaluated the roles of the M512 and V513 residues in M. tuberculosis AHAS. Interestingly, these residues also exhibited conserved interactions with herbicides in plant AHAS [16]. However, surprisingly, substitution of these residues did not greatly affect the inhibition kinetics. Specifically, the M512L and V513L point mutants showed only 4-fold and 2-fold higher IC50 values than the wild type, respectively. Also, docking studies revealed that the binding energies of point mutants were not significantly affected. Therefore, we conclude that the M512 and V513 residues constitute part of the herbicide-binding site in M. tuberculosis AHAS, but they do not interact directly with the herbicide. In conclusion, mutational analysis of multiple known herbicidebinding residues led to altered kinetic parameters and inhibition properties of M. tuberculosis AHAS by a well-known herbicide, CE. The altered kinetic parameters for various point mutants towards substrate and cofactors indicated that the selected residues might have some catalytic function in M. tuberculosis AHAS. R318 appears to be a particularly important residue, since point mutations of this residue resulted in a complete loss of enzymatic activity. In addition, the point mutants tested demonstrated significant resistance against CE compared with wild type AHAS, indicating that these residues might be involved in herbicide binding. Collectively, the results presented here suggest that the residues examined might play an important role in CE binding to AHAS and are also likely to be involved in the catalytic activity of AHAS.
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Fig. 12. Conformations of the R318 point mutants. The yellow CPK model indicates the following residues: (a) R318, (b) A318, (c) H318, and (d) E318. The stick model indicates ThDP.
Acknowledgement This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01133002)” funded by the Rural Development Administration. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.enzmictec.2015. 06.009 References [1] P.J. Dolin, M.C. Raviglione, A. Kochi, Global tuberculosis incidence and mortality during 1990–2000, Bull. World Health Organ. 72 (1994) 213–220. [2] D.H. Kim, H.J. Kim, S.K. Park, S.J. Kong, Y.S. Kim, T.H. Kim, E.K. Kim, K.M. Lee, S.S. Lee, J.S. Park, W.J. Koh, C.H. Lee, J.Y. Kim, T.S. Shim, Treatment outcomes and long-term survival in patients with extensively drug-resistant tuberculosis, Am. J. Respir. Crit. Care Med. 178 (2008) 1075–1082. [3] A. Zumla, P. Nahid, S.T. Cole, Advances in the development of new tuberculosis drugs and treatment regimens, Nat. Rev. Drug Discov. 12 (2013) 388–404. [4] N. Pue, L.W. Guddat, Acetohydroxyacid synthase: a target for antimicrobial drug discovery, Curr. Pharm. Des. (2014) 740–753. [5] F.C. Bange, A.M. Brown, W.R. Jacobs Jr, Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages, Infect. Immun. 64 (1996) 1794–1799. [6] I. Guleria, R. Teitelbaum, R.A. McAdam, G. Kalpana, W.R. Jacobs Jr, B.R. Bloom, Auxotrophic vaccines for tuberculosis, Nat. Med. 2 (1996) 334–337. [7] D. Chipman, Z. Barak, J.V. Schloss, Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases, Biochim. Biophys. Acta 1385 (1998) 401–419. [8] R.G. Duggleby, S.S. Pang, Acetohydroxyacid synthase, J. Biochem. Mol. Biol. 33 (2000) 1–36. [9] J.A. McCourt, R.G. Duggleby, Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids, Amino Acids 31 (2006) 173–210. [10] S.S. Pang, R.G. Duggleby, L.W. Guddat, Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors, J. Mol. Biol. 317 (2002) 249–262.
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