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Kate S. Carroll - One of the best experts on this subject based on the ideXlab platform.
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Design, synthesis and evaluation of Fe-S targeted Adenosine 5'-Phosphosulfate reductase inhibitors.
Nucleosides nucleotides & nucleic acids, 2015Co-Authors: Hanumantharao Paritala, Yuta Suzuki, Kate S. CarrollAbstract:Adenosine 5′-Phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of Mycobacterium tuberculosis during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, Adenosine 5′-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of N6-substituted Adenosine analogues and their 5′-phosphates as well as Adenosine 5′-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and Adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular doc...
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Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors
2015Co-Authors: Kate S. Carroll, Hanumantharao Paritala, Yuta SuzukiAbstract:Adenosine 5′-Phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of Mycobacterium tuberculosis during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, Adenosine 5′-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of N6-substituted Adenosine analogues and their 5′-phosphates as well as Adenosine 5′-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and Adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular docking analysis suggests that the S atom of the most potent inhibitor may establish a favorable interaction with an S atom in the cluster. In summary, this study showcases an improved solid-phase method that expedites the preparation of Adenosine and related 5′-phosphate derivatives and presents a unique Fe-S targeting strategy for the development of APR inhibitors.
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A continuous spectrophotometric assay for Adenosine 5'-Phosphosulfate reductase activity with sulfite-selective probes.
Analytical biochemistry, 2013Co-Authors: Hanumantharao Paritala, Kate S. CarrollAbstract:Abstract Mycobacterium tuberculosis (Mtb) Adenosine 5′-Phosphosulfate (APS) reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of essential reduced sulfur-containing biomolecules, such as cysteine, and is essential for survival in the latent phase of tuberculosis (TB) infection. Despite the importance of APR to Mtb and other bacterial pathogens, current assay methods depend on the use of 35S-labeled APS or shunt Adenosine 5′-monophosphate (AMP) to a coupled-enzyme system. Both methods are cumbersome and require the use of expensive reagents. Here, we report the development of a continuous spectrophotometric method for measuring APR activity by using novel sulfite-selective colorimetric or “off–on” fluorescent levulinate-based probes. Thus, the APR activity can be followed by monitoring the increase in absorbance or fluorescence of the resulting phenolate product. Using this assay, we determined Michaelis–Menten kinetic constants (Km, kcat, and kcat/Km) and the apparent inhibition constant (Ki) for Adenosine 5′-diphosphate (ADP), which compared favorably with values obtained in the “gold standard” radioactive assay. The newly developed assay is robust and easy to perform with a simple spectrophotometer.
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geometric and electrostatic study of the 4fe 4s cluster of Adenosine 5 Phosphosulfate reductase from broken symmetry density functional calculations and extended x ray absorption fine structure spectroscopy
Inorganic Chemistry, 2011Co-Authors: Devayani P. Bhave, Kate S. Carroll, Wen Ge Han, Samuel Pazicni, James E Pennerhahn, Louis NoodlemanAbstract:Adenosine-5′-Phosphosulfate reductase (APSR) is an iron–sulfur protein that catalyzes the reduction of Adenosine-5′-Phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X∼80-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron–sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S]2+ state in both native and substrate-bound APSR while EXAFS data recorded at ∼0.1 A resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On...
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Geometric and Electrostatic Study of the [4Fe-4S] Cluster of Adenosine-5′-Phosphosulfate Reductase from Broken Symmetry Density Functional Calculations and Extended X-ray Absorption Fine Structure Spectroscopy
Inorganic chemistry, 2011Co-Authors: Devayani P. Bhave, Kate S. Carroll, Wen Ge Han, Samuel Pazicni, James E. Penner-hahn, Louis NoodlemanAbstract:Adenosine-5′-Phosphosulfate reductase (APSR) is an iron–sulfur protein that catalyzes the reduction of Adenosine-5′-Phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X∼80-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron–sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S]2+ state in both native and substrate-bound APSR while EXAFS data recorded at ∼0.1 A resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On...
Stanislav Kopriva - One of the best experts on this subject based on the ideXlab platform.
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the x ray crystal structure of apr b an atypical Adenosine 5 Phosphosulfate reductase from physcomitrella patens
FEBS Letters, 2013Co-Authors: Clare E. M. Stevenson, Richard K. Hughes, Michael T. Mcmanus, David M. Lawson, Stanislav KoprivaAbstract:Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where Adenosine 5′-Phosphosulfate (APS) reductases possess a cluster and 3′-phosphoAdenosine 5′-Phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.
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The X-ray crystal structure of APR-B, an atypical Adenosine 5′-Phosphosulfate reductase from Physcomitrella patens
FEBS letters, 2013Co-Authors: Clare E. M. Stevenson, Richard K. Hughes, Michael T. Mcmanus, David M. Lawson, Stanislav KoprivaAbstract:Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where Adenosine 5′-Phosphosulfate (APS) reductases possess a cluster and 3′-phosphoAdenosine 5′-Phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.
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The key enzyme of sulfate assimilation, Adenosine 5'-Phosphosulfate reductase, is regulated by HY5 in Arabidopsis.
The Plant journal : for cell and molecular biology, 2011Co-Authors: Bok-rye Lee, Anna Koprivova, Stanislav KoprivaAbstract:Summary Plant sulfate assimilation is regulated by demand for reduced sulfur, as is its key enzyme, Adenosine 5′-Phosphosulfate reductase (APR). In a genetic screen for mutants lacking this regulation, we identified the bZIP transcription factor LONG HYPOCOTYL 5 (HY5) as a necessary component of the regulatory circuit. Regulation of APR activity by the inhibitor of glutathione synthesis, buthionine sulfoximine, or by the precursor of cysteine, O-acetylserine, was disrupted in the hy5 mutant. When dark-adapted plants were re-illuminated, the rapid induction of APR1 and APR2 mRNA levels was attenuated in hy5 seedlings, but APR3 regulation was not affected. Chromatin immunoprecipitation revealed that HY5 binds directly to the APR1 and APR2 promoters but not to the APR3 promoter. Accordingly, the regulation of APR1 and APR2 by O-acetylserine was disturbed in hy5 roots. HY5 is also important for the coordination of nitrogen and sulfur assimilation, as, unlike the wild-type, hy5 mutants do not undergo a reduction in sulfate uptake and APR activity during nitrogen starvation. Altogether, these data show that HY5 plays an important role in regulation of APR gene expression and plant sulfate assimilation.
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Adenosine 5 Phosphosulfate kinase is essential for arabidopsis viability
FEBS Letters, 2010Co-Authors: Sarah G. Mugford, Colette Matthewman, Lionel Hill, Stanislav KoprivaAbstract:In Arabidopsis thaliana, Adenosine-5'-Phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.
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Adenosine-5′-Phosphosulfate kinase is essential for Arabidopsis viability.
FEBS letters, 2009Co-Authors: Sarah G. Mugford, Colette Matthewman, Lionel Hill, Stanislav KoprivaAbstract:In Arabidopsis thaliana, Adenosine-5'-Phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.
Devayani P. Bhave - One of the best experts on this subject based on the ideXlab platform.
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geometric and electrostatic study of the 4fe 4s cluster of Adenosine 5 Phosphosulfate reductase from broken symmetry density functional calculations and extended x ray absorption fine structure spectroscopy
Inorganic Chemistry, 2011Co-Authors: Devayani P. Bhave, Kate S. Carroll, Wen Ge Han, Samuel Pazicni, James E Pennerhahn, Louis NoodlemanAbstract:Adenosine-5′-Phosphosulfate reductase (APSR) is an iron–sulfur protein that catalyzes the reduction of Adenosine-5′-Phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X∼80-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron–sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S]2+ state in both native and substrate-bound APSR while EXAFS data recorded at ∼0.1 A resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On...
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Geometric and Electrostatic Study of the [4Fe-4S] Cluster of Adenosine-5′-Phosphosulfate Reductase from Broken Symmetry Density Functional Calculations and Extended X-ray Absorption Fine Structure Spectroscopy
Inorganic chemistry, 2011Co-Authors: Devayani P. Bhave, Kate S. Carroll, Wen Ge Han, Samuel Pazicni, James E. Penner-hahn, Louis NoodlemanAbstract:Adenosine-5′-Phosphosulfate reductase (APSR) is an iron–sulfur protein that catalyzes the reduction of Adenosine-5′-Phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X∼80-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron–sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S]2+ state in both native and substrate-bound APSR while EXAFS data recorded at ∼0.1 A resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On...
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spectroscopic studies on the 4fe 4s cluster in Adenosine 5 Phosphosulfate reductase from mycobacterium tuberculosis
Journal of Biological Chemistry, 2011Co-Authors: Devayani P. Bhave, Jiyoung A. Hong, Kate S. Carroll, Michael Lee, Wei Jiang, Carsten KrebsAbstract:Mycobacterium tuberculosis Adenosine 5′-Phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S]2+ cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S]+ state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including Adenosine 5′-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of Adenosine 5′-Phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
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Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis
The Journal of biological chemistry, 2010Co-Authors: Devayani P. Bhave, Jiyoung A. Hong, Michael Lee, Wei Jiang, Carsten Krebs, Kate S. CarrollAbstract:Mycobacterium tuberculosis Adenosine 5′-Phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S]2+ cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S]+ state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including Adenosine 5′-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of Adenosine 5′-Phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
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Identification of Critical Ligand Binding Determinants in Mycobacterium tuberculosis Adenosine-5′-Phosphosulfate Reductase
Journal of medicinal chemistry, 2009Co-Authors: Jiyoung A. Hong, Devayani P. Bhave, Kate S. CarrollAbstract:Mycobacterium tuberculosis Adenosine-5'-Phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.
Jiyoung A. Hong - One of the best experts on this subject based on the ideXlab platform.
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Deciphering the role of histidine 252 in mycobacterial Adenosine 5'-Phosphosulfate (APS) reductase catalysis.
The Journal of biological chemistry, 2011Co-Authors: Jiyoung A. Hong, Kate S. CarrollAbstract:Mycobacterium tuberculosis Adenosine 5′-Phosphosulfate reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on Adenosine 5′-Phosphosulfate, resulting in a covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249 resides on a mobile active site lid at the C terminus, within a K(R/T)ECG(L/I)H motif. Owing to its strict conservation among sulfonucleotide reductases and its proximity to the active site cysteine, it has been suggested that His-252 plays a key role in APR catalysis, specifically as a general base to deprotonate Cys-249. Using site-directed mutagenesis, we have changed His-252 to an alanine residue and analyzed the effect of this mutation on the kinetic parameters, pH rate profile, and ionization of Cys-249 of APR. Interestingly, our data demonstrate that His-252 does not perturb the pKa of Cys-249 or play a direct role in rate-limiting chemical steps of the reaction. Rather, we show that His-252 enhances substrate affinity via interaction with the α-phosphate and the endocyclic ribose oxygen. These findings were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly His-252, may lead to inhibitors with improved binding affinity.
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spectroscopic studies on the 4fe 4s cluster in Adenosine 5 Phosphosulfate reductase from mycobacterium tuberculosis
Journal of Biological Chemistry, 2011Co-Authors: Devayani P. Bhave, Jiyoung A. Hong, Kate S. Carroll, Michael Lee, Wei Jiang, Carsten KrebsAbstract:Mycobacterium tuberculosis Adenosine 5′-Phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S]2+ cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S]+ state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including Adenosine 5′-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of Adenosine 5′-Phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
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Spectroscopic Studies on the [4Fe-4S] Cluster in Adenosine 5′-Phosphosulfate Reductase from Mycobacterium tuberculosis
The Journal of biological chemistry, 2010Co-Authors: Devayani P. Bhave, Jiyoung A. Hong, Michael Lee, Wei Jiang, Carsten Krebs, Kate S. CarrollAbstract:Mycobacterium tuberculosis Adenosine 5′-Phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S]2+ cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S]+ state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including Adenosine 5′-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of Adenosine 5′-Phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
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Identification of Critical Ligand Binding Determinants in Mycobacterium tuberculosis Adenosine-5′-Phosphosulfate Reductase
Journal of medicinal chemistry, 2009Co-Authors: Jiyoung A. Hong, Devayani P. Bhave, Kate S. CarrollAbstract:Mycobacterium tuberculosis Adenosine-5'-Phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.
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identification of critical ligand binding determinants in mycobacterium tuberculosis Adenosine 5 Phosphosulfate reductase
Journal of Medicinal Chemistry, 2009Co-Authors: Jiyoung A. Hong, Devayani P. Bhave, Kate S. CarrollAbstract:Mycobacterium tuberculosis Adenosine-5'-Phosphosulfate (APS) reductase is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. To facilitate the development of potent and specific inhibitors of APS reductase, we have probed the molecular determinants that underlie binding and specificity through a series of substrate and product analogues. Our study highlights the importance of specific substitutent groups for substrate binding and provides functional evidence for ligand-specific conformational states. An active site model has been developed for M. tuberculosis APS reductase that is in accord with the results presented here as well as prior structural data reported for Pseudomonas aeruginosa APS reductase and related enzymes. This model illustrates the functional features required for the interaction of APS reductase with a ligand and provides a pharmacological roadmap for the rational design of small molecules as potential inhibitors of APS reductase present in human pathogens, including M. tuberculosis.
Volker Schünemann - One of the best experts on this subject based on the ideXlab platform.
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The presence of an iron-sulfur cluster in Adenosine 5'-Phosphosulfate reductase separates organisms utilizing Adenosine 5'-Phosphosulfate and phosphoAdenosine 5'-Phosphosulfate for sulfate assimilation.
The Journal of biological chemistry, 2002Co-Authors: Stanislav Kopriva, Gunter Fritz, Thomas Buchert, Marianne Suter, Rüdiger Benda, Peter Schürmann, Volker Schünemann, Anna Koprivova, Alfred X. Trautwein, Peter M H KroneckAbstract:Abstract It was generally accepted that plants, algae, and phototrophic bacteria use Adenosine 5′-Phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoAdenosine 5′-Phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25–30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBankTM, revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those fromPseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacteriumPlectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. Mossbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.
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plant Adenosine 5 Phosphosulfate reductase is a novel iron sulfur protein
Journal of Biological Chemistry, 2001Co-Authors: Stanislav Kopriva, Gunter Fritz, Thomas Buchert, Markus Weber, Rüdiger Benda, Johann Schaller, Urs Feller, Peter Schürmann, M Suter, Volker SchünemannAbstract:Abstract Adenosine 5′-Phosphosulfate reductase (APR) catalyzes the two-electron reduction of Adenosine 5′-Phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minorand Arabidopsis thaliana were overexpressed inEscherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mossbauer spectra of 57Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S]2+ cluster. This cluster was unusual because only three of the iron sites exhibited the same Mossbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of Adenosine 5′-Phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory Adenosine 5′-Phosphosulfate reductases found in sulfate reducing bacteria.
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Plant Adenosine 5′-Phosphosulfate Reductase Is a Novel Iron-Sulfur Protein
The Journal of biological chemistry, 2001Co-Authors: Stanislav Kopriva, Gunter Fritz, Thomas Buchert, Marianne Suter, Markus Weber, Rüdiger Benda, Johann Schaller, Urs Feller, Peter Schürmann, Volker SchünemannAbstract:Abstract Adenosine 5′-Phosphosulfate reductase (APR) catalyzes the two-electron reduction of Adenosine 5′-Phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minorand Arabidopsis thaliana were overexpressed inEscherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mossbauer spectra of 57Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S]2+ cluster. This cluster was unusual because only three of the iron sites exhibited the same Mossbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of Adenosine 5′-Phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory Adenosine 5′-Phosphosulfate reductases found in sulfate reducing bacteria.