The project proposes an investigation of the control of synthesis, assimilation and recycling of the central metabolic cofactors, NAD and NADP. Emphasis is on pursuing preliminary evidence that cells protect themselves from oxidative stress by reducing their NADH pool. This minimizes production of the damaging -OH radical from H2O2 via the nonenzymatic Fenton reaction, which requires Fe+2 and a reducing agent. NADH, but not NADPH, can serve as the required reducing agent. By reducing NAD levels temporarily, cells can 'freeze' metabolism, allowing time for DNA repair and destruction of H2O2. The model proposes that NAD levels are reduced by conversion of NAD to NADP, followed by destruction of excess NADP. The NMN produced may be excreted to the periplasm for later uptake when growth resumes. The second product of NAD(P) breakdown, 2' 5'ADP, may play a regulatory role in control of the response to oxidative stress. Control of internal iron levels may also be involved in the stress responses; one of the NAD kinases (NAD-more than NADP) has already been shown to be activated by Fe. Other enzymes that initiate the NAD cycle may also be controlled by either O2 or Fe. It is not clear how cells control the relative size of their NAD and NADP pools. We will sequence the two NAD kinase genes, make lac fusions and expression plasmids. Using the fusions, the transcriptional regulation of these genes will be pursued. The purified enzymes will be analysed for modulation of their activity in response to oxidative stress, iron and pyridine precursor limitation. The essential NMN deamidase maintains a low internal level of the toxic intermediate, NMN; we will study the control of this step. Excess NMN may be excreted by the pnuC transporter which appears to contribute to both import and excretion of NMN. The energetics of this transporter will be investigated to determine whether NAD serves to dictate the direction of flow. We will continue the search for transporters of Nm or Na have been described. It seems likely that there must be some control on assimilation of these compound. Several candidates for Nm and Na transporters are in hand which will be pursued to determine how many transporters exist and whether any are controlled. The nadD gene will be tested as a likely candidate for a step that might be regulated to control assimilation and recycling. This compound is a synthetic intermediate, the entry point of assimilated Nm and Na, the entry point of recycled NMN and the source of ribose for synthesis of cobalamin. A missense suppressor will be investigated that may act by unbalancing rNTP pools and increasing the transcriptional error frequency. We pursue this because it is part of a novel idea regarding a possible chemotherapy for AIDS. The idea involves increasing the error rate in transcriptional replication of HIV by attacking host targets, which control purine and pyrimidine pool sizes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023408-21
Application #
2021764
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-01-01
Project End
1998-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
21
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Utah
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Li, Chan; Edwards, Michelle D; Jeong, Hotcherl et al. (2007) Identification of mutations that alter the gating of the Escherichia coli mechanosensitive channel protein, MscK. Mol Microbiol 64:560-74
Grose, Julianne H; Joss, Lisa; Velick, Sidney F et al. (2006) Evidence that feedback inhibition of NAD kinase controls responses to oxidative stress. Proc Natl Acad Sci U S A 103:7601-6
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Grose, Julianne H; Bergthorsson, Ulfar; Xu, Yaping et al. (2005) Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica. J Bacteriol 187:4521-30
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Zhu, N; Olivera, B M; Roth, J R (1989) Genetic characterization of the pnuC gene, which encodes a component of the nicotinamide mononucleotide transport system in Salmonella typhimurium. J Bacteriol 171:4402-9

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