Structural and Functional Studies of Nicotinamide Adenine Dinucleotide and Streptolysin S Biosynthesis Proteins from Streptococcus pyogenes .

摘要

Invasive infections caused by Streptococcus pyogenes , also known as Group A Strep (GAS), results in approximately 600,000 deaths annually. With evidence of antibiotic-resistant strains of this bacterium on the rise, there is a need for the identification of new drug targets to control these infections. In our approach we target the quinolinate-salvage pathway (QSP) and the streptolysin S (SLS) biosynthesis pathway. The QSP provides a secondary pathway for NAD+ biosynthesis within this organism; the SLS pathway leads to the formation of a quorum sensing molecule (SLS). We hypothesize that inhibition of the pathways will lead to GAS cell death or will impair the growth of the bacterium.;This dissertation presents results of the functional and structural (using x-ray crystallography) characterization of select proteins involved in these pathways. Highlights of the information contained within each chapter are as follows: (1) Chapter 1 provides background information on the QSP and SLS pathways and reports current research on both targets. (2) Chaper 2 describes results from structural studies on quinolinate phosphoribosyltransferase (spNadC), of the QSP, and shows results from functional and structural studies, as well as the first structures of this protein and its deletion mutant (spNadC Delta69A). (3) Chapter 3 reports the results from functional characterization of nicotinate mononucleotide adenyltransferase (spNadD) of the QSP. Preliminary structural studies are described. While the protein structure was not yet determined, homology modeling was used to provide insights into the proposed spNadD structure. (4) Chapter 4 details the functional and structural studies on NAD+ synthetase (spNadE) of the QSP. This protein, similarly to NadD, is considered to be a good drug target. This chapter describes the first structures of this protein determined in GAS. Structural results provide new details for the reaction mechanism and the conversion of the substrate (nicotinate adenine dinucleotide (NaAD)) into nicotinamide adenine dinucleotide (NAD+) through channels identified at the dimer interface of spNadE. (5) Chapter 5 describes results from molecular cloning, isolation, and refolding experiments conducted on select SLS biosynthesis proteins: SagB, SagC, SagD, and SagG. Structures of these proteins have not been determined; however, molecular cloning experiments for the co-expression of the SagBCD complex have shown promising results.