Reducing the Mysteries of Sulfur Metabolism in Mycobacterium Tuberculosis.

Sulfur metabolic pathways are fundamental for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. However, many aspects of mycobacterial sulfur metabolism, such as mechanistic details of sulfonucleotide reductases (SRs) involved in assimilatory sulfate reduction, remain poorly understood and represent exciting areas of new or continued investigation. SRs catalyze the first committed step of reductive sulfur assimilation en route to the biosynthesis of all sulfur-containing metabolites. In this study, we elucidate the molecular binding determinants that underlie ligand binding and specificity of SRs and provide a pharmacological roadmap for the rational design of potential inhibitors of SRs. Next, we present a spectroscopic characterization of the iron-sulfur cofactor essential to one class of SRs and reveal mid-range electrostatic interactions between the iron-sulfur cluster and the substrate in the active site. Based on these data, we propose a role for the cluster in pre-organizing active site residues and in substrate activation. Computational modeling and theoretical calculations corroborate these findings and in addition, suggest a role for the unique coordination of the iron-sulfur cluster in facilitating a compact geometric structure and modulating its electrostatic nature. Furthermore, metalloprotein engineering, kinetic and spectroscopic analyses demonstrate that the iron-sulfur cluster plays a pivotal role in substrate specificity and catalysis, and yield important structural information that can be used for the design of cluster-targeted SR inhibitors. The findings also provide new perspectives into the evolution of the SR family, and have broader implications regarding the function of protein-bound iron-sulfur clusters. Collectively, the work presented in this thesis contributes towards a better understanding of the catalytic mechanism of this unique class of enzymes and offers insights into strategies for therapeutic intervention.Ph.D.Chemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89843/1/devayani_1.pd

A 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-X80-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 Å 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 the other hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APS-bound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and “fine-tuning” the electronic structure to prevent reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS

Iron–Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among Sulfonucleotide Reductases

Assimilatory sulfate reduction supplies prototrophic organisms with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation <i>via</i> an ATP-dependent activation to form adenosine-5′-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes have no human homologue, rendering them attractive targets for the development of novel antibacterial drugs. APR and PAPR share sequence and structure homology as well as a common catalytic mechanism, but the enzymes are distinguished by two features, namely, the amino acid sequence of the phosphate-binding loop (P-loop) and an iron–sulfur cofactor in APRs. On the basis of the crystal structures of APR and PAPR, two P-loop residues are proposed to determine substrate specificity; however, this hypothesis has not been tested. In contrast to this prevailing view, we report here that the P-loop motif has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic, and kinetic analyses, we demonstrate that the iron–sulfur cluster cofactor enhances APS reduction by nearly 1000-fold, thereby playing a pivotal role in substrate specificity and catalysis. These findings offer new insights into the evolution of this enzyme family and extend the known functions of protein-bound iron–sulfur clusters