Control of pyridine nucleotide synthesis and recycling in Salmonella enterica.

摘要

This dissertation describes the regulation of pyridine levels in the bacterium S. enterica. More specifically, it describes the regulation of total pyridine levels by the trifunctional enzyme NadR, and the regulation of the NAD to NADP ratio by NAD kinase.;This study describes the first genetic and biochemical characterization of S. enterica NAD kinase (encoded by nadK). In contrast to previous reports of multiple NAD kinases, nadK was shown to be essential; consistent with its role as the only producer of NADP. The Km's of this enzyme for NAD and ATP are comparable to the in vivo concentrations of these compounds, making NAD kinase sensitive to slight variations in these levels. NAD kinase is potently inhibited by NADPH both in vitro and in vivo, allowing the cell to maintain a high NADPH to NADP ratio that is optimal for biosynthesis. Furthermore, NAD kinase is hypothesized to play a role in oxidative stress by increasing NADP levels within the cell as NADPH levels drop due to oxidative repair processes.;The trifunctional protein NadR was characterized and shown to posses NmR kinase activity, along with previously reported DNA binding and NMNAT activities. Genetic and biochemical evidence is presented supporting two independent conformations of NadR: an NAD-induced repressor conformation and an enzymatically active (NMNAT and NmRK) conformation. Thus, NadR regulates the pyridine levels within the cell by repressing NAD biosynthetic genes (nadA, nadB, pncB, pnuC) when NAD levels are high, and assimilating NmR when NAD levels are limiting.;NAD and NADP are essential cofactors, making identification of enzymes involved in their biosynthesis difficult. In an attempt to identify new NAD-related genes, NAD pathways were reconstructed in different organisms by searching for homologues of known NAD genes. In addition, the Functional Clustering program was used to identify genes that are frequently near NAD biosynthetic genes. These genes are most likely involved in NAD metabolism due to the frequency of clustered functionally-related genes in bacterial genomes.