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Design, synthesis and evaluation of Fe-S targeted Adenosine 5‘-Phosphosulfate reductase inhibitors.

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…

Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors

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.

A continuous spectrophotometric assay for Adenosine 5‘-Phosphosulfate reductase activity with sulfite-selective probes.

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.

the x ray crystal structure of apr b an atypical Adenosine 5 Phosphosulfate reductase from physcomitrella patens

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.

The X-ray crystal structure of APR-B, an atypical Adenosine 5′-Phosphosulfate reductase from Physcomitrella patens

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.

The key enzyme of sulfate assimilation, Adenosine 5‘-Phosphosulfate reductase, is regulated by HY5 in Arabidopsis.

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.

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

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…

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

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…

spectroscopic studies on the 4fe 4s cluster in Adenosine 5 Phosphosulfate reductase from mycobacterium tuberculosis

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.