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Dinesh Christendat - One of the best experts on this subject based on the ideXlab platform.
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the Shikimate Dehydrogenase family functional diversity within a conserved structural and mechanistic framework
Archives of Biochemistry and Biophysics, 2015Co-Authors: James Peek, Dinesh ChristendatAbstract:Shikimate Dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-deydroShikimate to Shikimate, an essential reaction in the biosynthesis of the aromatic amino acids and a large number of other secondary metabolites in plants and microbes. The indispensible nature of this enzyme makes it a potential target for herbicides and antimicrobials. SDH is the archetypal member of a large protein family, which contains at least four additional functional classes with diverse metabolic roles. The different members of the SDH family share a highly similar three-dimensional structure and utilize a conserved catalytic mechanism, but exhibit distinct substrate preferences, making the family a particularly interesting system for studying modes of substrate recognition used by enzymes. Here, we review our current understanding of the biochemical and structural properties of each of the five previously identified SDH family functional classes.
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identification of novel polyphenolic inhibitors of Shikimate Dehydrogenase aroe
Journal of Biomolecular Screening, 2014Co-Authors: James Peek, Thomas Shi, Dinesh ChristendatAbstract:Shikimate Dehydrogenase (AroE) is an attractive target for herbicides and antimicrobial agents due to its conserved and essential nature in plants, fungi, and bacteria. Here, we have performed an in vitro screen using a collection of more than 5500 compounds and identified 24 novel inhibitors of AroE from Pseudomonas putida The IC50 values for the two most potent inhibitors we identified, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), were 3.0 ± 0.2 µM and 3.7 ± 0.5 µM, respectively. Based on the high level of structural conservation between AroE orthologs, we predicted that the identified compounds would also inhibit AroE enzymes from other organisms. Consistent with this hypothesis, we found that EGCG and ECG inhibit the AroE domain of the bifunctional dehydroquinate dehydratase-Shikimate Dehydrogenase (DHQ-SDH) from Arabidopsis thaliana with IC50 values of 2.1 ± 0.3 µM and 2.0 ± 0.2 µM, respectively.
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isolation and molecular characterization of the Shikimate Dehydrogenase domain from the toxoplasma gondii arom complex
Molecular and Biochemical Parasitology, 2014Co-Authors: James Peek, Thomas Shi, Gianni M Castiglione, Dinesh ChristendatAbstract:The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10-80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the Shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the Shikimate Dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP(+)-dependent oxidation of Shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the 'core' structure of the enzyme.
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insights into the function of rifi2 structural and biochemical investigation of a new Shikimate Dehydrogenase family protein
Biochimica et Biophysica Acta, 2013Co-Authors: James Peek, Dinesh Christendat, Christel Garcia, John LeeAbstract:Abstract The Shikimate Dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using Shikimate as a substrate than AroE, and a strong preference for NAD+ instead of NADP+ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD+ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD+-dependent member of the SDH family.
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Structural and Mechanistic Analysis of a Novel Class of Shikimate Dehydrogenases: Evidence for a Conserved Catalytic Mechanism in the Shikimate Dehydrogenase Family
Biochemistry, 2011Co-Authors: James Peek, John G. Lee, Guillermo Senisterra, Dinesh ChristendatAbstract:Shikimate Dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroShikimate to Shikimate. This reaction represents the fourth step of the Shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize Shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH h...
James Peek - One of the best experts on this subject based on the ideXlab platform.
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the Shikimate Dehydrogenase family functional diversity within a conserved structural and mechanistic framework
Archives of Biochemistry and Biophysics, 2015Co-Authors: James Peek, Dinesh ChristendatAbstract:Shikimate Dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-deydroShikimate to Shikimate, an essential reaction in the biosynthesis of the aromatic amino acids and a large number of other secondary metabolites in plants and microbes. The indispensible nature of this enzyme makes it a potential target for herbicides and antimicrobials. SDH is the archetypal member of a large protein family, which contains at least four additional functional classes with diverse metabolic roles. The different members of the SDH family share a highly similar three-dimensional structure and utilize a conserved catalytic mechanism, but exhibit distinct substrate preferences, making the family a particularly interesting system for studying modes of substrate recognition used by enzymes. Here, we review our current understanding of the biochemical and structural properties of each of the five previously identified SDH family functional classes.
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identification of novel polyphenolic inhibitors of Shikimate Dehydrogenase aroe
Journal of Biomolecular Screening, 2014Co-Authors: James Peek, Thomas Shi, Dinesh ChristendatAbstract:Shikimate Dehydrogenase (AroE) is an attractive target for herbicides and antimicrobial agents due to its conserved and essential nature in plants, fungi, and bacteria. Here, we have performed an in vitro screen using a collection of more than 5500 compounds and identified 24 novel inhibitors of AroE from Pseudomonas putida The IC50 values for the two most potent inhibitors we identified, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), were 3.0 ± 0.2 µM and 3.7 ± 0.5 µM, respectively. Based on the high level of structural conservation between AroE orthologs, we predicted that the identified compounds would also inhibit AroE enzymes from other organisms. Consistent with this hypothesis, we found that EGCG and ECG inhibit the AroE domain of the bifunctional dehydroquinate dehydratase-Shikimate Dehydrogenase (DHQ-SDH) from Arabidopsis thaliana with IC50 values of 2.1 ± 0.3 µM and 2.0 ± 0.2 µM, respectively.
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isolation and molecular characterization of the Shikimate Dehydrogenase domain from the toxoplasma gondii arom complex
Molecular and Biochemical Parasitology, 2014Co-Authors: James Peek, Thomas Shi, Gianni M Castiglione, Dinesh ChristendatAbstract:The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10-80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the Shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the Shikimate Dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP(+)-dependent oxidation of Shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the 'core' structure of the enzyme.
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insights into the function of rifi2 structural and biochemical investigation of a new Shikimate Dehydrogenase family protein
Biochimica et Biophysica Acta, 2013Co-Authors: James Peek, Dinesh Christendat, Christel Garcia, John LeeAbstract:Abstract The Shikimate Dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using Shikimate as a substrate than AroE, and a strong preference for NAD+ instead of NADP+ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD+ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD+-dependent member of the SDH family.
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Structural and Mechanistic Analysis of a Novel Class of Shikimate Dehydrogenases: Evidence for a Conserved Catalytic Mechanism in the Shikimate Dehydrogenase Family
Biochemistry, 2011Co-Authors: James Peek, John G. Lee, Guillermo Senisterra, Dinesh ChristendatAbstract:Shikimate Dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroShikimate to Shikimate. This reaction represents the fourth step of the Shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize Shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH h...
Diogenes Santiago Santos - One of the best experts on this subject based on the ideXlab platform.
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the conserved lysine69 residue plays a catalytic role in mycobacterium tuberculosis Shikimate Dehydrogenase
BMC Research Notes, 2009Co-Authors: Valnes Da Silva Rodrigues, Ardala Breda, Diogenes Santiago Santos, Luiz Augusto BassoAbstract:The Shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded Shikimate Dehydrogenase catalyzes the forth reaction in the Shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis Shikimate Dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis Shikimate Dehydrogenase. We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis Shikimate Dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis Shikimate Dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM) and NADPH (K69A = 30 μM; wild-type = 11 μM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4) μM and 134 (± 21), respectively. Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis Shikimate Dehydrogenase. These efforts on M. tuberculosis Shikimate Dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.
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homogeneous recombinant mycobacterium tuberculosis Shikimate Dehydrogenase production an essential step towards target based drug design
International Journal of Biological Macromolecules, 2009Co-Authors: Valnes Rodriguesjunior, Luiz Augusto Basso, Diogenes Santiago SantosAbstract:Mycobacterium tuberculosis Shikimate Dehydrogenase (MtbSD) catalyzes the forth reaction in the Shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.
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structural studies of Shikimate 5 Dehydrogenase from mycobacterium tuberculosis
Proteins, 2008Co-Authors: Helen Andrade Arcuri, Diogenes Santiago Santos, Luiz Augusto Basso, Isabel Osorio Da Fonseca, Julio Cesar Borges, Jose Henrique Pereira, Joao Ruggiero Neto, Walter Filgueira De AzevedoAbstract:Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the Shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial Shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the Shikimate Dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the Shikimate pathway. Our structural models show that the MtSDH has similar structure to other Shikimate Dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50°C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of ∼63–64°C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents. Proteins 2008. © 2008 Wiley-Liss, Inc.
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kinetic and chemical mechanisms of Shikimate Dehydrogenase from mycobacterium tuberculosis
Archives of Biochemistry and Biophysics, 2007Co-Authors: Isabel Osorio Da Fonseca, Luiz Augusto Basso, Rafael G Silva, Claudia Lemelle Fernandes, Osmar Norberto De Souza, Diogenes Santiago SantosAbstract:Mycobacterium tuberculosis Shikimate Dehydrogenase (MtbSD) catalyzes the fourth reaction in the Shikimate pathway, the NADPH-dependent reduction of 3-dehydroShikimate. To gather information on the kinetic mechanism, initial velocity patterns, product inhibition, and primary deuterium kinetic isotope effect studies were performed and the results suggested a steady-state ordered bi-bi kinetic mechanism. The magnitudes of both primary and solvent kinetic isotope effects indicated that the hydride transferred from NADPH and protons transferred from the solvent in the catalytic cycle are not significantly rate limiting in the overall reaction. Proton inventory analysis indicates that one proton gives rise to solvent isotope effects. Multiple isotope effect studies indicate that both hydride and proton transfers are concerted. The pH profiles revealed that acid/base chemistry takes place in catalysis and substrate binding. The MtbSD 3D model was obtained in silico by homology modeling. Kinetic and chemical mechanisms for MtbSD are proposed on the basis of experimental data.
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functional Shikimate Dehydrogenase from mycobacterium tuberculosis h37rv purification and characterization
Protein Expression and Purification, 2006Co-Authors: Isabel Osorio Da Fonseca, Diogenes Santiago Santos, Maria L B Magalhaes, Jaim S Oliveira, Rafael G Silva, Maria Anita Mendes, Mario Sergio Palma, Luiz Augusto BassoAbstract:Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth Shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded Shikimate Dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis Shikimate Dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.
Luiz Augusto Basso - One of the best experts on this subject based on the ideXlab platform.
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the conserved lysine69 residue plays a catalytic role in mycobacterium tuberculosis Shikimate Dehydrogenase
BMC Research Notes, 2009Co-Authors: Valnes Da Silva Rodrigues, Ardala Breda, Diogenes Santiago Santos, Luiz Augusto BassoAbstract:The Shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded Shikimate Dehydrogenase catalyzes the forth reaction in the Shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis Shikimate Dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis Shikimate Dehydrogenase. We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis Shikimate Dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis Shikimate Dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM) and NADPH (K69A = 30 μM; wild-type = 11 μM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4) μM and 134 (± 21), respectively. Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis Shikimate Dehydrogenase. These efforts on M. tuberculosis Shikimate Dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.
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homogeneous recombinant mycobacterium tuberculosis Shikimate Dehydrogenase production an essential step towards target based drug design
International Journal of Biological Macromolecules, 2009Co-Authors: Valnes Rodriguesjunior, Luiz Augusto Basso, Diogenes Santiago SantosAbstract:Mycobacterium tuberculosis Shikimate Dehydrogenase (MtbSD) catalyzes the forth reaction in the Shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.
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structural studies of Shikimate 5 Dehydrogenase from mycobacterium tuberculosis
Proteins, 2008Co-Authors: Helen Andrade Arcuri, Diogenes Santiago Santos, Luiz Augusto Basso, Isabel Osorio Da Fonseca, Julio Cesar Borges, Jose Henrique Pereira, Joao Ruggiero Neto, Walter Filgueira De AzevedoAbstract:Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the Shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial Shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the Shikimate Dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the Shikimate pathway. Our structural models show that the MtSDH has similar structure to other Shikimate Dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50°C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of ∼63–64°C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents. Proteins 2008. © 2008 Wiley-Liss, Inc.
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kinetic and chemical mechanisms of Shikimate Dehydrogenase from mycobacterium tuberculosis
Archives of Biochemistry and Biophysics, 2007Co-Authors: Isabel Osorio Da Fonseca, Luiz Augusto Basso, Rafael G Silva, Claudia Lemelle Fernandes, Osmar Norberto De Souza, Diogenes Santiago SantosAbstract:Mycobacterium tuberculosis Shikimate Dehydrogenase (MtbSD) catalyzes the fourth reaction in the Shikimate pathway, the NADPH-dependent reduction of 3-dehydroShikimate. To gather information on the kinetic mechanism, initial velocity patterns, product inhibition, and primary deuterium kinetic isotope effect studies were performed and the results suggested a steady-state ordered bi-bi kinetic mechanism. The magnitudes of both primary and solvent kinetic isotope effects indicated that the hydride transferred from NADPH and protons transferred from the solvent in the catalytic cycle are not significantly rate limiting in the overall reaction. Proton inventory analysis indicates that one proton gives rise to solvent isotope effects. Multiple isotope effect studies indicate that both hydride and proton transfers are concerted. The pH profiles revealed that acid/base chemistry takes place in catalysis and substrate binding. The MtbSD 3D model was obtained in silico by homology modeling. Kinetic and chemical mechanisms for MtbSD are proposed on the basis of experimental data.
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functional Shikimate Dehydrogenase from mycobacterium tuberculosis h37rv purification and characterization
Protein Expression and Purification, 2006Co-Authors: Isabel Osorio Da Fonseca, Diogenes Santiago Santos, Maria L B Magalhaes, Jaim S Oliveira, Rafael G Silva, Maria Anita Mendes, Mario Sergio Palma, Luiz Augusto BassoAbstract:Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth Shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded Shikimate Dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis Shikimate Dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.
Sasha A. Singh - One of the best experts on this subject based on the ideXlab platform.
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the dhq dehydroShikimate sdh Shikimate nadp h complex insights into metabolite transfer in the Shikimate pathway
Crystal Growth & Design, 2007Co-Authors: Sasha A. Singh, Dinesh ChristendatAbstract:Plants encode the bifunctional dehydroquinase-Shikimate Dehydrogenase (DHQ-SDH), which catalyzes the third and fourth steps of the Shikimate pathway. We report the Arabidopsis thaliana DHQ-SDH structure in complex with all of its natural substrates. The DHQ-SDH enzyme was first cocrystallized with Shikimate. NADP+ was subsequently added to the crystals yielding the SDH ternary complex. The Pro-R hydrogen of the nicotinamide C4 is 3.35 A from the C3 of Shikimate, the site of hydride transfer. The catalytic Lys 385 and Asp 423 residues are proximal to the C3-hydroxyl of Shikimate, which is deprotonated in the oxidation reaction. The SDH-Shikimate-NADP(H) complex represents the active complex as the oxidation of Shikimate was evidenced by the generation of the product (dehydroShikimate) found in the DHQ site. DHQ-SDH adopts a concave architecture that places the active sites in a face-to-face arrangement. This proximal organization serves to increase the local effective concentration of dehydroShikimate, and...
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Structure of Arabidopsis dehydroquinate dehydratase-Shikimate Dehydrogenase and implications for metabolic channeling in the Shikimate pathway.
Biochemistry, 2006Co-Authors: Sasha A. Singh, Dinesh ChristendatAbstract:The bifunctional enzyme dehydroquinate dehydratase-Shikimate Dehydrogenase (DHQ-SDH) catalyzes the dehydration of dehydroquinate to dehydroShikimate and the reduction of dehydroShikimate to Shikimate in the Shikimate pathway. We report the first crystal structure of Arabidopsis DHQ-SDH with Shikimate bound at the SDH site and tartrate at the DHQ site. The interactions observed in the DHQ-tartrate complex reveal a conserved mode for substrate binding between the plant and microbial DHQ dehydratase family of enzymes. The SDH-Shikimate complex provides the first direct evidence of the role of active site residues in the catalytic mechanism. Site-directed mutagenesis and mechanistic analysis revealed that Asp 423 and Lys 385 are key catalytic groups and Ser 336 is a key binding group. The arrangement of the two functional domains reveals that the control of metabolic flux through the Shikimate pathway is achieved by increasing the effective concentration of dehydroShikimate through the proximity of the two sites.
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crystal structure of a novel Shikimate Dehydrogenase from haemophilus influenzae
Journal of Biological Chemistry, 2005Co-Authors: Sasha A. Singh, Sergey Korolev, Olga Koroleva, T. I. Zarembinski, Frank R. Collart, Andrzej Joachimiak, Dinesh ChristendatAbstract:To date two classes of Shikimate Dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroShikimate to Shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroShikimate to Shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of Shikimate Dehydrogenase annotated as Shikimate Dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of Shikimate but not quinate. The turnover rate for the oxidation of Shikimate is ∼1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of Shikimate Dehydrogenases, namely AroE, YdiB, and this newly characterized Shikimate Dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the Shikimate Dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of Shikimate Dehydrogenase.
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Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae
Journal of Biological Chemistry, 2005Co-Authors: Sasha A. Singh, Sergey Korolev, Olga Koroleva, T. I. Zarembinski, Frank R. Collart, Andrzej Joachimiak, Dinesh ChristendatAbstract:To date two classes of Shikimate Dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroShikimate to Shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroShikimate to Shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of Shikimate Dehydrogenase annotated as Shikimate Dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of Shikimate but not quinate. The turnover rate for the oxidation of Shikimate is approximately 1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of Shikimate Dehydrogenases, namely AroE, YdiB, and this newly characterized Shikimate Dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the Shikimate Dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of Shikimate Dehydrogenase.