Mechanistic analysis of essential enzymes from mycobacterium tuberculosis and other bacterial pathogens

Abstract

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), an infectious disease accounting for nearly two million deaths annually. The increasing prevalence of drug resistant TB and other bacterial organisms has exacerbated the need for novel antibiotics. Kinetic and mechanistic analysis of essential enzymes in pathogenic bacteria will provide insight for rational drug discovery and deepen our understanding of bacterial metabolism.;beta-Lactamase (B1aC) is the enzyme responsible for the intrinsic resistance to beta-lactam antibiotics in M tuberculosis. We have determined solvent kinetic isotope effects for three beta-lactam substrates (nitrocefin, cefoxitin, and meropenem) whose kcat values differ by 4.5 orders of magnitude. The rate-limiting steps for beta-lactam hydrolysis were analyzed and the chemical steps responsible for the observed solvent kinetic isotope effects are discussed.;Nicotinate phosphoribosyltransferase (NAPRT, PncB) catalyzes the first step of the NAD+ salvage pathway. The two homologs that encode NAPRT in M tuberculosis are believed to be activated during TB persistence. As its chemical mechanism is amenable to transition state inhibitor design, NAPRT may be a potential bactericidal target against latent and drug resistant tuberculosis. We have attempted to characterize the kinetic mechanism of NAPRT and assess its potential for inhibitor design.;L-Aspartate oxidase is the first enzyme in the de novo synthesis of NAD+ in bacteria. Distinct from most amino acid oxidases, it can use either oxygen or fumarate to re-oxidize the reduced enzyme. Crystal structures have revealed that the folding topology of NadB closely resembles that of the succinate dehydrogenase/fumarate reductase rather than the prototypical D-amino acid oxidases. We have investigated this mechanistic ambiguity using deuterium kinetic isotope effects. Together with previous data, we propose that NadB has structurally evolved from SDH/FRD enzymes to gain the new functionality of oxidizing amino acids.;Distinct from humans, M tuberculosis bacilli utilize the MEP pathway for the synthesis of isoprenoids. The first committed step of this pathway is catalyzed by 1deoxy-D-xylulose 5-phosphate reductoisomerase (DXR). Using DNA-Encoded Library Technology, we have identified and evaluated two micromolar inhibitors against M tuberculosis DXR. Our studies suggest that the enzyme utilizes an ordered sequential mechanism with NADPH binding first, followed by DXP binding.