Reducing the Mysteries of Sulfur Metabolism in Mycobacterium Tuberculosis.

Abstract

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.