Enzyme and Microbial Technology 58–59 (2014) 52–59
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Structural and functional significance of the highly-conserved residues in Mycobacterium tuberculosis acetohydroxyacid synthase Irshad Ahmed Baig a , Ji-Young Moon a , Min-Seo Kim a , Bon-Sung Koo b , Moon-Young Yoon a,∗ a b
Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133791, Republic of Korea Department of Agricultural Biotechnology, National Academy of Agricultural Science, Suwon 441707, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 5 February 2014 Accepted 18 February 2014 Available online 28 February 2014 Keywords: Acetohydroxyacid synthase ThDP dependent enzymes Mutagenesis Molecular modeling Molecular dynamics
a b s t r a c t Mycobacterium tuberculosis AHAS is a potential target for the development of novel anti-tuberculosis agents. Silico analysis showed that conserved His84 and Gln86 residues lie in the catalytic dimer interface of M. tuberculosis AHAS. Mutational analyses of these invariants led to significant reduction in their activity with reduced affinity toward the substrate. Importantly, mutation of Gln86 to Trp abolished complete activity. Further, molecular dynamics simulation studies suggested that these residues are likely to play a key role in maintaining the Glu85 side chain in the required geometry with N1 atom of ThDP during catalysis. In addition, substitution of essential Glu85 by Ala, Asp, and Gln led to severe drop in catalytic activity with reduced affinity toward ThDP confirming its catalytic role in M. tuberculosis AHAS. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis, has remained one of the leading causes of death worldwide. The World Health Organization currently estimated that 1.8 billion people are infected with M. tuberculosis, and around 2 million deaths each year because of tuberculosis [1]. This current situation demands the development of more effective anti-tuberculosis agents based on new potential targets. Acetohydroxyacid synthase (AHAS) is essential for the growth of M. tuberculosis [2] and has recently gained attention as a promising anti-tuberculosis drug target [3–5]. Earlier studies proved that the BCAA-auxotrophic strains of M. tuberculosis fail to proliferate in their hosts due to their inability to use amino acids from their hosts [6]. Further, absence of AHAS in animals, made AHAS as an attractive target for antimicrobials, antifungal agents and herbicides [7–9]. Hence, a deeper understanding of the structure of M. tuberculosis AHAS would help in designing and discovering more potent
Abbreviations: M. tuberculosis AHAS, Mycobacterium tuberculosis acetohydroxyacid synthase; CSU, catalytic subunit; ThDP, thiamine diphosphate; BCAA, branched-chain amino acids; MD, molecular dynamics. ∗ Corresponding author. Tel.: +82 2 2220 0946; fax: +82 2 2299 0763. E-mail addresses: [email protected] (I.A. Baig), [email protected] (J.-Y. Moon), [email protected] (M.-S. Kim), [email protected] (B.-S. Koo), [email protected] (M.-Y. Yoon). http://dx.doi.org/10.1016/j.enzmictec.2014.02.009 0141-0229/© 2014 Elsevier Inc. All rights reserved.
inhibitors. However, no crystal structure of AHAS from M. tuberculosis is currently available. While pursuit of a crystal structure is continued, a site directed mutagenesis study would be a useful tool to gain some insights about the important residues involved in catalysis and inhibition. Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase) catalyzes the first step in the biosynthesis of branched-chain amino acids (BCAAs) such as isoleucine, leucine, and valine [10]. This reaction involves synthesizing either (2S)-acetolactate (AL) from two molecules of pyruvate or (2S)-2aceto-2-hydroxybutyrate (AHB) from pyruvate and 2-ketobutyrate (KB). AL and AHB are obligatory intermediates in valine and isoleucine biosynthesis, respectively [11]. AHAS requires a thiamine diphosphate cofactor (ThDP, also known as thiamine pyrophosphate), a divalent metal ion such as Mg2+ to anchor ThDP to the enzyme, and a molecule of flavin adenine dinucleotide (FAD). AHAS is comprised of two subunits: a large catalytic subunit and a small regulatory one. The catalytic subunit (CSU) has a molecular mass of 60–70 kDa and is active alone. The role of the conserved Glu residue in plant and yeast AHAS, were studied and supported by several mutagenesis experiments previously [12–16]. According to these studies the presence of N1 atom of the ThDP and the Glu residue is crucial for the catalytic activity of AHAS and it is suggested that their interaction is important in the very first step in catalysis, deprotonation of the thiazolium ring at the C-2 atom. Several studies so far have focused on residues lying in the binding pocket of the cofactor ThDP, however only few studies have addressed the role
I.A. Baig et al. / Enzyme and Microbial Technology 58–59 (2014) 52–59
53
Table 1 Primers used for PCR and sequencing. S. No.
Variants
Primers
1. 2. 3. 4. 5. 6. 7.
E85Q E85D E85A H84A H84T Q86A Q86W
5 CAC GTG CTG GTC CGC CAC CAG CAG GGC GCC GGG CAT GCC GCC3 5 CAC GTG CTG GTC CGC CAC GAC CAG GGC GCC GGG CAT GCC GCC3 5 CAC GTG CTG GTC CGC CAC GCC CAG GGC GCC GGG CAT GCC GCC3 5 GTG CTG GTC CGC GCA GAA CAG GGC GCC GGG3 5 CAC GTG CTG GTC CGC ACC GAA CAG GGC GCC3 5 GTC CGC CAC GAA GCA GGC GCC GGG CAT3 5 CTG GTC CGC CAC GAA TGG GGC GCC GGG CAT3
of other conserved residues present in the immediate proximity of the catalytic glutamate [17]. Briefly, this study demonstrates the identification of catalytic Glu85 and further suggests the key supporting role of adjacent conserved H84 and Qln86 in M. tuberculosis AHAS.
2.3. Expression and purification of wild-type and variants The genes encoding wild type and variant M. tuberculosis AHAS catalytic subunits were cloned into the pET28a vector and expressed in competent E. coli BL21-DE3. The growth of cells, expression, and purification of AHAS protein (wild type and variants) were based on previously described methods [27].
2. Materials and methods 2.1. Multiple sequence alignment, homology modeling, and dynamics simulation The complete protein sequence of AHAS CSU of M. tuberculosis H37Rv (TIGR locus: NT02MT3272) was retrieved from the Comprehensive Microbial Resource of the J. Craig Venter Institute (http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi) and aligned with AHAS from 28 different species using ClustalW2 [18]. The set of all organisms used for alignment and comparison of AHAS is provided in supporting information S1. For computational studies, the homology modeling of M. tuberculosis AHAS was performed by using 1N0H and 1JSC (yeast AHAS) as template as previously [19] and the best model was parameterized according to GROMOS96 43a1 force field [20] for performing molecular dynamics simulation in the GROMACS 4.5.6 [21]. The PRODRG server [22] was used to generate topology files of ligands. The model was solvated in a cubic water box extending 1.2 nanometer (nm) from the surface of the structure with single point charge (SPC216) water model [23] followed by the neutralization by adding 20 Sodium ions to the system. Energy minimization was performed using the steepest descent for 50,000 steps and restricted to the maximum force
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Structural and functional significance of the highly-conserved residues in Mycobacterium tuberculosis acetohydroxyacid synthase Irshad Ahmed Baig a , Ji-Young Moon a , Min-Seo Kim a , Bon-Sung Koo b , Moon-Young Yoon a,∗ a b
Department of Chemistry, Institute of Natural Science, Hanyang University, Seoul 133791, Republic of Korea Department of Agricultural Biotechnology, National Academy of Agricultural Science, Suwon 441707, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 5 February 2014 Accepted 18 February 2014 Available online 28 February 2014 Keywords: Acetohydroxyacid synthase ThDP dependent enzymes Mutagenesis Molecular modeling Molecular dynamics
a b s t r a c t Mycobacterium tuberculosis AHAS is a potential target for the development of novel anti-tuberculosis agents. Silico analysis showed that conserved His84 and Gln86 residues lie in the catalytic dimer interface of M. tuberculosis AHAS. Mutational analyses of these invariants led to significant reduction in their activity with reduced affinity toward the substrate. Importantly, mutation of Gln86 to Trp abolished complete activity. Further, molecular dynamics simulation studies suggested that these residues are likely to play a key role in maintaining the Glu85 side chain in the required geometry with N1 atom of ThDP during catalysis. In addition, substitution of essential Glu85 by Ala, Asp, and Gln led to severe drop in catalytic activity with reduced affinity toward ThDP confirming its catalytic role in M. tuberculosis AHAS. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Tuberculosis (TB), caused by Mycobacterium tuberculosis, has remained one of the leading causes of death worldwide. The World Health Organization currently estimated that 1.8 billion people are infected with M. tuberculosis, and around 2 million deaths each year because of tuberculosis [1]. This current situation demands the development of more effective anti-tuberculosis agents based on new potential targets. Acetohydroxyacid synthase (AHAS) is essential for the growth of M. tuberculosis [2] and has recently gained attention as a promising anti-tuberculosis drug target [3–5]. Earlier studies proved that the BCAA-auxotrophic strains of M. tuberculosis fail to proliferate in their hosts due to their inability to use amino acids from their hosts [6]. Further, absence of AHAS in animals, made AHAS as an attractive target for antimicrobials, antifungal agents and herbicides [7–9]. Hence, a deeper understanding of the structure of M. tuberculosis AHAS would help in designing and discovering more potent
Abbreviations: M. tuberculosis AHAS, Mycobacterium tuberculosis acetohydroxyacid synthase; CSU, catalytic subunit; ThDP, thiamine diphosphate; BCAA, branched-chain amino acids; MD, molecular dynamics. ∗ Corresponding author. Tel.: +82 2 2220 0946; fax: +82 2 2299 0763. E-mail addresses: [email protected] (I.A. Baig), [email protected] (J.-Y. Moon), [email protected] (M.-S. Kim), [email protected] (B.-S. Koo), [email protected] (M.-Y. Yoon). http://dx.doi.org/10.1016/j.enzmictec.2014.02.009 0141-0229/© 2014 Elsevier Inc. All rights reserved.
inhibitors. However, no crystal structure of AHAS from M. tuberculosis is currently available. While pursuit of a crystal structure is continued, a site directed mutagenesis study would be a useful tool to gain some insights about the important residues involved in catalysis and inhibition. Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase) catalyzes the first step in the biosynthesis of branched-chain amino acids (BCAAs) such as isoleucine, leucine, and valine [10]. This reaction involves synthesizing either (2S)-acetolactate (AL) from two molecules of pyruvate or (2S)-2aceto-2-hydroxybutyrate (AHB) from pyruvate and 2-ketobutyrate (KB). AL and AHB are obligatory intermediates in valine and isoleucine biosynthesis, respectively [11]. AHAS requires a thiamine diphosphate cofactor (ThDP, also known as thiamine pyrophosphate), a divalent metal ion such as Mg2+ to anchor ThDP to the enzyme, and a molecule of flavin adenine dinucleotide (FAD). AHAS is comprised of two subunits: a large catalytic subunit and a small regulatory one. The catalytic subunit (CSU) has a molecular mass of 60–70 kDa and is active alone. The role of the conserved Glu residue in plant and yeast AHAS, were studied and supported by several mutagenesis experiments previously [12–16]. According to these studies the presence of N1 atom of the ThDP and the Glu residue is crucial for the catalytic activity of AHAS and it is suggested that their interaction is important in the very first step in catalysis, deprotonation of the thiazolium ring at the C-2 atom. Several studies so far have focused on residues lying in the binding pocket of the cofactor ThDP, however only few studies have addressed the role
I.A. Baig et al. / Enzyme and Microbial Technology 58–59 (2014) 52–59
53
Table 1 Primers used for PCR and sequencing. S. No.
Variants
Primers
1. 2. 3. 4. 5. 6. 7.
E85Q E85D E85A H84A H84T Q86A Q86W
5 CAC GTG CTG GTC CGC CAC CAG CAG GGC GCC GGG CAT GCC GCC3 5 CAC GTG CTG GTC CGC CAC GAC CAG GGC GCC GGG CAT GCC GCC3 5 CAC GTG CTG GTC CGC CAC GCC CAG GGC GCC GGG CAT GCC GCC3 5 GTG CTG GTC CGC GCA GAA CAG GGC GCC GGG3 5 CAC GTG CTG GTC CGC ACC GAA CAG GGC GCC3 5 GTC CGC CAC GAA GCA GGC GCC GGG CAT3 5 CTG GTC CGC CAC GAA TGG GGC GCC GGG CAT3
of other conserved residues present in the immediate proximity of the catalytic glutamate [17]. Briefly, this study demonstrates the identification of catalytic Glu85 and further suggests the key supporting role of adjacent conserved H84 and Qln86 in M. tuberculosis AHAS.
2.3. Expression and purification of wild-type and variants The genes encoding wild type and variant M. tuberculosis AHAS catalytic subunits were cloned into the pET28a vector and expressed in competent E. coli BL21-DE3. The growth of cells, expression, and purification of AHAS protein (wild type and variants) were based on previously described methods [27].
2. Materials and methods 2.1. Multiple sequence alignment, homology modeling, and dynamics simulation The complete protein sequence of AHAS CSU of M. tuberculosis H37Rv (TIGR locus: NT02MT3272) was retrieved from the Comprehensive Microbial Resource of the J. Craig Venter Institute (http://cmr.jcvi.org/cgi-bin/CMR/CmrHomePage.cgi) and aligned with AHAS from 28 different species using ClustalW2 [18]. The set of all organisms used for alignment and comparison of AHAS is provided in supporting information S1. For computational studies, the homology modeling of M. tuberculosis AHAS was performed by using 1N0H and 1JSC (yeast AHAS) as template as previously [19] and the best model was parameterized according to GROMOS96 43a1 force field [20] for performing molecular dynamics simulation in the GROMACS 4.5.6 [21]. The PRODRG server [22] was used to generate topology files of ligands. The model was solvated in a cubic water box extending 1.2 nanometer (nm) from the surface of the structure with single point charge (SPC216) water model [23] followed by the neutralization by adding 20 Sodium ions to the system. Energy minimization was performed using the steepest descent for 50,000 steps and restricted to the maximum force
